Integrative Bayesian Analysis Identifies Rhabdomyosarcoma Disease Genes

Article

Integrative Bayesian Analysis Identifies Rhabdomyosarcoma Disease Genes Graphical Abstract

Authors Lin Xu, Yanbin Zheng, Jing Liu, ..., Douglas S. Hawkins, James F. Amatruda, Stephen X. Skapek

Correspondence [email protected] (Y.Z.), [email protected] (S.X.S.)

In Brief Xu et al. use an integrative computational pipeline (iExCN) to identify 25 candidate oncogenic driver genes and 4 tumor suppressors in rhabdomyosarcoma, and many are validated in a CRISPR/ Cas9 mini-pool screen. Functional assays and correlation with survival indicate that cooperative interactions across iExCN genes contribute to disease biology, including differentiation arrest.

Highlights d

Integrative analysis (iExCN) reveals candidate RMS driver and suppressor genes

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CRISPR/Cas9 mini-pool screen validates many iExCN candidates in cultured RMS lines

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Multiple iExCN drivers contribute to RMS growth and differentiation arrest

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A higher number of iExCN genes with copy-number alteration correlates with poor survival

Xu et al., 2018, Cell Reports 24, 238–251 July 3, 2018 ª 2018 The Authors. https://doi.org/10.1016/j.celrep.2018.06.006

Data and Software Availability GSE114621

Cell Reports

Article Integrative Bayesian Analysis Identifies Rhabdomyosarcoma Disease Genes Lin Xu,1,2,13 Yanbin Zheng,1,13,* Jing Liu,1 Dinesh Rakheja,3,5 Sydney Singleterry,1 Theodore W. Laetsch,1,5,6 Jack F. Shern,7 Javed Khan,8 Timothy J. Triche,9,10 Douglas S. Hawkins,11,12 James F. Amatruda,1,4,5,6 and Stephen X. Skapek1,5,6,14,* 1Department

of Pediatrics, Division of Hematology/Oncology, University of Texas Southwestern Medical Center, Dallas, TX of Clinical Sciences, University of Texas Southwestern Medical Center, Dallas, TX 3Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 4Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 5Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 6Gill Center for Cancer and Blood Disorders, Children’s Health, Children’s Medical Center, Dallas, TX 7Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 8Oncogenomics Section, Genetic Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 9Department of Pathology, Keck School of Medicine at USC, Los Angeles, CA 10Department of Pediatrics, Keck School of Medicine at USC, Los Angeles, CA 11Division of Pediatric Hematology/Oncology, Seattle Children’s Hospital, Seattle, WA 12Fred Hutchinson Cancer Research Center, University of Washington, Seattle, WA 13These authors contributed equally 14Lead Contact *Correspondence: [email protected] (Y.Z.), [email protected] (S.X.S.) https://doi.org/10.1016/j.celrep.2018.06.006 2Department

SUMMARY

Identifying oncogenic drivers and tumor suppressors remains a challenge in many forms of cancer, including rhabdomyosarcoma. Anticipating gene expression alterations resulting from DNA copy-number variants to be particularly important, we developed a computational and experimental strategy incorporating a Bayesian algorithm and CRISPR/ Cas9 ‘‘mini-pool’’ screen that enables both genomescale assessment of disease genes and functional validation. The algorithm, called iExCN, identified 29 rhabdomyosarcoma drivers and suppressors enriched for cell-cycle and nucleic-acid-binding activities. Functional studies showed that many iExCN genes represent rhabdomyosarcoma line-specific or shared vulnerabilities. Complementary experiments addressed modes of action and demonstrated coordinated repression of multiple iExCN genes during skeletal muscle differentiation. Analysis of two separate cohorts revealed that the number of iExCN genes harboring copy-number alterations correlates with survival. Our findings highlight rhabdomyosarcoma as a cancer in which multiple drivers influence disease biology and demonstrate a generalizable capacity for iExCN to unmask disease genes in cancer. INTRODUCTION In the genomics era, technological advancements and sophisticated computational approaches to identify mutated genes have illuminated the molecular basis of many cancer hallmarks (Hana-

han and Weinberg, 2011), but rarely has that insight reached the clinic. The shortcoming is variably due to the paucity or the vast number of somatic single nucleotide variants (SNVs) in a tumor, limited recurrence of specific SNVs within an individual cancer type, lack of ability to target a cancer-driving mutation, and incorrect assumptions about the relevance of individual aberrations (Heuckmann and Thomas, 2015). This problem applies to rhabdomyosarcoma (RMS), an aggressive soft tissue sarcoma composed of skeletal myoblast-like cells (Hettmer and Wagers, 2010; Saab et al., 2011). The cancer is divided into two major histologic subtypes marked by embryonal or alveolar histology features (ERMS and ARMS, respectively), with most ARMS carrying a balanced translocation that generates an oncogenic fusion protein consisting of PAX3 or PAX7 and FOXO1 (Saab et al., 2011). Next-generation sequencing approaches reveal that the fusion-positive (FP) RMS cases harbor few additional chromosomal rearrangements and somatic SNVs; in contrast, fusionnegative (FN) cases display many more SNVs, but recurrence of SNVs across RMS specimens is very limited (Chen et al., 2013; Seki et al., 2015; Shern et al., 2014). In contrast to SNVs, DNA copy-number variations (CNVs) are common and more highly recurrent in FN RMS (Paulson et al., 2011). Therefore, we considered that altered expression of key genes may be ‘‘hard wired’’ by coordinately increased or decreased copies of those genes. To sift through the vast number of CNVs, we developed a pipeline incorporating Bayesian methodology (Kruschke, 2013; Kruschke and Liddell, 2018) to integrate expression and copy number (iExCN pipeline). The iExCN analysis of FN RMS revealed candidate oncogenic drivers and tumor suppressor genes (TSGs), most of which were not previously known to be important in this disease. We used a ‘‘mini-pool’’ of lentiviral CRISPR/Cas9 vectors and additional functional analyses to validate the importance of many iExCN-defined genes. We revealed multiple genes with coordinate low-level

238 Cell Reports 24, 238–251, July 3, 2018 ª 2018 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Figure 1. Integrative Genomic Analysis Identifies Few Recurrent SNVs and Frequent Copy-Number Alterations in RMS (A) Venn diagrams display the number of RMS cases and type of genomic data used in this analysis. (B) Analysis of somatic protein-altering single nucleotide variants (SNVs) in 134 pairs of RMS tumor-normal specimens with whole-genome sequencing (WGS) data, whole-exome sequencing (WES) data, or both reveals that the vast majority of >3,827 mutated genes were found only in single RMS cases. (C) Charts show statistically significant regions displaying copy-number gains (left) and losses (right) identified by applying the Genomic Identification of Significant Targets in Cancer 2.0 (GISTIC2) algorithm in RMS cases with PAX3/7-FOXO1 fusion genes (FP cases) or without fusion genes (FN cases). See also Tables S1, S2, S3, and S4.

copy-number and expression changes that contribute to RMS, illuminated mechanisms underlying a corrupted myogenic differentiation program, and identified potentially actionable therapeutic targets and a prognostic biomarker for this disease. Here, we provide a general approach to identify oncogenic drivers and TSGs in forms of cancer in which mutation calling and CNV analyses have failed to reveal the full spectrum of disease drivers. RESULTS The iExCN Pipeline Reveals 29 Candidate RMS Oncogenic Drivers and Tumor Suppressors Using published (Shern et al., 2014) and unpublished data derived from 290 RMS specimens (Figure 1A; Table S1), we

confirmed recent reports showing the paucity of recurrent SNVs in most protein-coding genes (Chen et al., 2013; Seki et al., 2015; Shern et al., 2014). For example, in 134 FN and FP RMS cases with whole-genome sequencing (WGS) or wholeexome sequencing (WES) data, we identified somatic proteinaltering SNVs in 3,827 genes, with 3,034 (79%) found in only single cases and 3,748 (98%) identified in three or fewer different cases (Figure 1B; Table S2). Mutated genes with recurrence in three or more cases included NF1, KRAS, NRAS, PIK3CA, and FGFR4, again highlighting the importance of RTK/RAS/PIK3CA signaling in RMS (Chen et al., 2013; Seki et al., 2015; Shern et al., 2014). Given the limited SNV recurrence, we considered whether integrative analyses of CNVs could provide a more complete

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description of cancer drivers and suppressors. Genomic Identification of Significant Targets in Cancer (GISTIC) analysis demonstrated many CNVs in both RMS forms, with many more (n = 1,797) significantly altered genes in FN RMS (Figure 1C; Table S3), consistent with previous reports (Chen et al., 2013; Seki et al., 2015; Shern et al., 2014). Considering that functionally relevant CNVs would likely influence the expression of the involved protein-coding genes, we developed iExCN based on Bayesian estimation of the probability that the expression of individual genes is correlated with CNVs (Figure S1A). (Details of the development and testing and a link to iExCN source code are provided in the Supplemental Experimental Procedures and Figure S1). Focusing on FN RMS, which harbors many more CNVs, we used iExCN to uncover coordinate protein-coding gene copy-number and expression changes. This analysis revealed 25 candidate oncogenic drivers and 4 tumor suppressors, assuming that copy-number gain and increased expression signified oncogenes and vice versa for TSGs (Figure 2A). Unlike SNVs that usually applied to only single FN RMS cases (Figure 1B), most iExCN genes were broadly expressed in FN RMS (Figure 2B). Similarly, the copy-number gains and losses in iExCN genes each involved R50 FN RMS cases in our cohort (Figures 2C and 2D). Functional Categories of iExCN Genes Eight (28%) of the 29 candidates were previously linked to RMS (CCND2, CDK6, EZH2, GLI2, GLI3, KIF18A, PTEN, and RIPK2) (Chen et al., 2013; Ehlers et al., 2008; Hatley et al., 2012; Li et al., 2007; Marchesi et al., 2012; Saab et al., 2006; Shern et al., 2014), and 28% are listed in the Catalogue of Somatic Mutations in Cancer (COSMIC) Cancer Gene Census (CCND2, CDK6, ETV1, EZH2, PLAG1, PTEN, WRN, and ZRSR2). This represents a significant enrichment when compared to the 3% found when sets of 29 genes were chosen randomly from (1) all protein-coding genes expressed in FN RMS (binomial test; p < 104), or (2) the 1,797 genes identified by GISTIC to harbor CNVs (binomial test; p < 104). Of note, 15 of the iExCN genes were among the 1,797 GISTIC genes (Table S4). We also note that six iExCN genes (CCND2, CDK6, GLI2, HAS2, SLC6A15, and ZFHX4) harbor PAX3-FOXO1 binding elements identified in previous chromatin immunoprecipitation sequencing (ChIPseq) studies (Cao et al., 2010; Gryder et al., 2017). Finally, 9 of the 29 iExCN genes (CCND2, CDK6, HAS2, KIF18A, RAD54B, SLC6A15, TJP2, TRIP13, and ZFHX4) contained somatic missense SNVs in 134 RMS cases. Each mutation was found in only a single case, except for TJP2 and TRIP13, which were mutated in two and three cases, respectively. To our knowledge, the significance of these rare SNVs remains to be determined. Only two Gene Ontology (GO) terms were significantly enriched in the iExCN-defined genes (Figure 2A). The largest category, nucleic acid binding, encompassed 12 of the 29 genes (false discovery rate [FDR]-adjusted p = 0.01), including transcriptional regulators related to the cell division cycle (E2F5), Hedgehog signaling (GLI2 and GLI3), histone modification (EZH2), epithelial-to-mesenchymal transition (SNAI2), and insulin-like growth factor 2 (IGF2) signaling (PLAG1). The next-largest Gene Ontology term, cell cycle, contained five genes (FDRadjusted p = 0.04), including those influencing the G1/S phase

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transition (CCND2, CDK6, and E2F5) and associated with microtubule formation and dynamics (KIF18A). Because transcription factor regulation and cell-cycle arrest are intimately linked to skeletal muscle differentiation, enrichment of those pathways underlines how RMS represents a cancer with derailed developmental programs (Hettmer and Wagers, 2010; Saab et al., 2011). CRISPR/Cas9 Mini-pool Screen Validates iExCNDefined Genes in RMS We verified the functional importance of iExCN genes using a competitive assay with a mini-pool of CRISPR/Cas9 lentiviral vectors targeting (1) iExCN genes, (2) negative control genes chosen randomly, and (3) positive controls, which were previously shown to be essential (Shalem et al., 2014) (Figure 3A; Tables S5 and S6). The iExCN genes and the negative and positive controls were expressed in most FN RMS cases (Figures 2B, 3B, and 3C). Following transduction and serial passage of two authenticated FN RMS lines (JR1 and RD) (Figures S2A and S2B), lentiviral vector DNA was analyzed by targeted deep sequencing to assess relative changes in vector representation (Figures 3A and S2C). In aggregate, the normalized mean read depth for vectors targeting positive control genes decreased by 17% and 20% in JR1 and RD cells (two-tailed Student’s t test; p = 2 3 10
6 and 2 3 10
8, respectively) between days 0 and 14. In contrast, representation of vectors targeting negative control genes did not change significantly (0.4% and 0.1% decrease in JR1 and RD [two-tailed Student’s t test; p = 0.84 and 0.95, respectively]), confirming the expected assay performance. We evaluated changes in the representation of lentiviral vectors targeting individual iExCN-derived and control genes. As in previous reports (Plaisier et al., 2016; Toledo et al., 2015), we defined significantly altered vectors as those meeting both statistical (FDR-corrected p < 0.05 by two-tailed Student’s t test) and magnitude of change (R20%) thresholds (Figures 3D–3F, asterisks and black lines, respectively). Only 2 of 51 vectors targeting negative controls were depleted, and none were enriched; in contrast, 17 of 30 vectors targeting positive controls were depleted in one or both of the tested RMS lines (Figures 3D, 3E, and 3G). Vectors targeting 15 of 29 iExCN-defined genes changed: 12 of 25 targeting candidate oncogenic drivers were depleted, and 3 of 4 targeting candidate TSGs were enriched (Figures 3F–3H). Non-specific toxicity mediated by CRISPR/Cas9 targeting genes with copy-number amplification has been reported by several groups (Aguirre et al., 2016; Munoz et al., 2016; Wang et al., 2015), and this raises the concern that certain of the 12 oncogenes identified as line-specific vulnerabilities in the mini-pool screen (Figure 3H) may include false positives due to gene copynumber gain. To address this, we first examined copy numbers of the iExCN and the positive control genes in the two RMS models JR1 and RD. We found that 75% (JR1) and 67% (RD) of CRISPR/Cas9 hits in iExCN genes and >80% of the hits in positive control genes lacked measurable copy-number amplification in these particular cell lines (Table S7). Furthermore, small interfering RNA (siRNA) knock down of each of the iExCNdefined oncogenic drivers impaired cell accumulation in the relevant lines (Figures 5A–5F and S3). Taken together, our data indicate that more than half of the iExCN genes represent true

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Figure 2. iExCN Analysis Identifies RMS Oncogenic Drivers and Tumor Suppressors (A) Map shows 29 candidate RMS oncogenic drivers and tumor suppressors (+) within Gene Ontology terms (shaded ovals). Physical and genetic interactions from the GeneMANIA database are indicated by solid and dashed lines, respectively. Cell-cycle and nucleic acid binding are the only categories with statistically significant enrichment. (B) Chart displays the number of FN RMS cases (%) in which each of 29 candidate RMS drivers or tumor suppressors has an expression level (fragments per kilobase of transcript per million mapped reads [FPKM]) of R1 based on RNA sequencing. (C) Charts display the average copy-number level for copy-number gain events of iExCN-predicted oncogenes and copy-number loss events of iExCN-predicted tumor suppressor genes (TSGs). (D) Charts display the number among 209 FN RMS cases with copy-number gain (red) and loss (blue) events on 29 iExCN-defined RMS disease genes. Data presented as mean ± SEM. See also Figure S1.

shared or line-specific vulnerabilities in the tested FN RMS models. Functional Interplay between iExCN Genes and Muscle Differentiation RMS is composed of myoblast-like cells in which the capacity for terminal differentiation is corrupted, but mechanisms underlying that differentiation defect are incompletely understood (Saab

et al., 2011). Hypothesizing that iExCN-predicted drivers may impair differentiation, we investigated whether they were differentially expressed in myoblasts versus differentiated myocytes. Analysis of human skeletal myoblasts propagated in vitro showed enhanced expression of muscle differentiation markers in myoblasts cultivated in differentiation-inducing medium (Figure 4A, red curves), while the expression of 14 (48%) of all 29 iExCN-predicted genes (56% of 25 candidate oncogenic drivers)

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negatively correlated with differentiation (Pearson correlation coefficients <
0.5) (Figure 4A, blue curves). In contrast, expression of the four TSGs did not associate significantly with differentiation (Figure 4A, green curves). Iteratively testing sets of 29 genes randomly selected from (1) all genes expressed in FN RMS (Figure 4B, gray bars) or (2) the 1,797 genes identified by GISTIC (Figure S4A, gray bars) revealed that most genes display little expression change with muscle differentiation (Pearson correlation coefficients
0.4 to 0.4). In both cases, finding expression of 48% of iExCN genes to fall with differentiation (Pearson correlation coefficient %
0.5) significantly exceeded the 15% found by chance (binomial test; p < 10
6) (Figures 4B and S4A, blue bars). Because no part of the iExCN pipeline filtered candidates based on expression changes during muscle differentiation, this finding further illustrates the potential for iExCN to identify biologically relevant genes. To extend our findings beyond mere correlation, we addressed mechanisms by which the differentially regulated iExCN genes fall with differentiation and how those genes can blunt muscle gene expression. Because a number of microRNA (miRNA) species have been recognized to play a role in coordinating the myoblast-to-myocyte transition (Ge and Chen, 2011), we investigated whether the decreased expression could be explained by miRNA-dependent targeting. After predicting miRNA binding sites in 30 UTR regions, we found 633 miRNAs that could target at least 1 iExCN gene, whereas each iExCN gene had 4–85 predicted miRNA partners (Figure S4B). The majority (492 of 633) of these miRNAs, however, were predicted to regulate only 1 iExCN gene, and no miRNA was predicted to regulate >5 (Figure S4C). These findings indicate that the coordinated effects of just a few miRNAs known to influence myogenesis are not likely to account for the differential expression of the iExCN genes. We then focused on several histone marks that are associated with active transcription (ENCODE Project Consortium, 2012). Analyzing DNA located within 5 kb of the transcription start sites for the 14 iExCN genes of interest, we observed that nearly all of them showed significant enrichment for H3K4me3, H3K27ac, and H3K9ac (Figures 4A, 4C, and S5). In all of them, the histone methylation and acetylation fell with differentiation, even for genes such as PTN, with relatively low expression and histone marks at baseline (Figures 4A and 4C). Hence, histone remodeling coordinates repression of these oncogenic drivers in muscle differentiation. High expression of certain iExCN drivers in myoblasts suggested that they may limit the transcriptional activity of MyoD,

a lineage-defining myogenic transcription factor (Saab et al., 2011) in myoblasts and RMS cells. Using a system in which MyoD activity is monitored by a reporter driven by enhancer or promoter elements from the CKM gene (Wilson et al., 2016), we found MyoD activity to be significantly blunted by co-expression of EZH2, PTN, SNAI2, and RIPK2 (Figure 4D). To directly test whether those drivers suppressed muscle gene expression in RMS, we studied how their transient knockdown influenced the expression of early and later markers of muscle differentiation in two FN (RD and RH18) lines and one FP (RH28) line (Figures 4E, 5C, 5E, and S6). Focusing on the expression of MYOG, a direct transcriptional target of MyoD (Saab et al., 2011) and an early step in the myoblast-to-myocyte transition (Andre´s and Walsh, 1996), we observed that knock down of each gene enhanced MYOG expression in one or more of the cell lines, revealing cell line-specific effects of individual iExCN genes (Figure 4E). The induction of later markers of differentiation was less consistent across the lines (Figure S6), highlighting the need for additional feed-forward pathways beyond MyoD activation for terminal differentiation (Penn et al., 2004). Viewed together, our findings indicate that deregulated expression of multiple genes with low-level copy-number gains may act cooperatively to impose a differentiation block in RMS. iExCN Oncogene Drivers as Potential Therapeutic Targets in RMS We used siRNA knockdown to confirm the importance of several of the iExCN drivers depleted in the CRISPR/Cas9 mini-pool: ZFHX4, SNAI2, and RIPK2. Transiently targeting each decreased the native transcript and significantly diminished cell accumulation in three FN RMS lines and two FP RMS lines (RH28 and RH30) (two-tailed Student’s t test; p < 0.05) (Figures 5A–5F). Hence, although FP and FN RMS are initiated by different mechanisms (Hettmer and Wagers, 2010; Saab et al., 2011), their shared myogenic lineage may lead to shared cooperating drivers or tumor suppressors. We focused further mechanistic and pharmacological studies on RIPK2 because it represents a targetable vulnerability in RMS (Canning et al., 2015). RIPK2 was previously implicated in myogenesis, but the mechanisms by which it acted were not defined (Munz et al., 2002). We tested whether it influenced the cyclin D/ cyclin-dependent kinase (CDK)/retinoblastoma (RB) axis because deregulated cyclin D1 blunts MyoD activity (Skapek et al., 1995), RIPK2 expression enhances nuclear factor kB (NF-kB) signaling (McCarthy et al., 1998), and NF-kB induces cyclin D1 (Guttridge et al., 1999). Indeed, RIPK2 knockdown in

Figure 3. iExCN-Predicted Disease Genes Are Validated by CRISPR/Cas9 Mini-pool Screen (A) Schematic diagram shows experimental protocol in which RMS cells are transduced with lentiviral vectors affecting CRISPR/Cas9-based targeting of candidate oncogenes and tumors suppressors as well as randomly chosen genes. Relative changes in lentivirus representation are measured by targeted deepsequencing at the beginning and the end of the experiment. (B and C) Charts display the number of FN RMS cases (%) in which each gene has an expression level (FPKM) of R1 based on RNA sequencing for negative (B) and positive (C) control genes. (D–F) Charts display average fold change of read counts of six sgRNAs per gene from days 0–14 for negative control (D), positive control (E), and iExCN genes (F). Asterisks represent FDR-corrected p < 0.05 by two-tailed Student’s t test. Black lines represent 20% increase or decrease. Data are presented for two RMS cell lines, JR1 and RD, as indicated. (G) Summary statistics for significantly altered genes targeted by depleted and enriched lentiviral vectors in the screen. (H) Venn diagram shows genes targeted by vectors that are significantly depleted or enriched (orange text) in the two FN RMS lines (JR1 and RD). Data presented as mean ± SD from six replicates. See also Figures S2 and S3 and Tables S5, S6, and S7.

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three RMS lines diminished CDK4/6-dependent phosphorylation of RB serine 780 (Figure 5G) and increased the expression of the CDKN1A gene product, p21CIP1, a CDK inhibitor induced in differentiating myocytes (Figures 5G and 5H). Ectopic RIPK2 also blocked the expression of an artificial enhancer reporter driven by reiterated MyoD-specific E-boxes (Skapek et al., 1995) (Figure S7A), demonstrating that RIPK2 expression interferes with transcriptional activation by MyoD or related transcription factors. We next considered whether pharmacological strategies to manipulate RIPK2 similarly control RMS accumulation and foster muscle gene expression. We focused on two compounds: PP1, previously shown to inhibit RIPK2 (Bain et al., 2007), and ponatinib, which blocks RIPK2 and other kinases (Canning et al., 2015). RIPK2 knockdown and exposure to PP1 or ponatinib consistently decreased cell accumulation in all tested RMS lines (Figures 5E, 5F, and 5I), while RIPK2 knockdown also augmented MYOG expression in RH18 and RH28 cells (Figure 4E). These findings align with another report in a panel of RMS models in vitro and in vivo (Li et al., 2013).

mosomal instability (CIN) index score, which positively correlates with genomic instability (Wang et al., 2013), we found that RMS cases with CNVs in a greater number of iExCN genes actually had a slightly lower CIN index score in one cohort (Figure 6E). Because that association was not confirmed in a second, independent cohort (Figure 6F), it should be interpreted cautiously. Second, simulations calculating the association between survival and CNVs in randomly selected 29-gene sets revealed that <1% of 10,000 iterations had a significant association (log-rank test; p < 0.05) in both cohorts. Finally, cases with CNVs involving >10 iExCN genes had significantly higher average expression of the 25 oncogenes (Figures 6G and 6H) (two-tailed Student’s t test; p < 0.05 for each) and a trend toward lower expression of the 4 candidate TSGs (Figure S7B). Hence, the prognostic value of CNVs involving iExCN genes did not stem from genome-wide copy number change per se; it depended on the identity of the genes involved and correlated with higher expression of the encoded drivers.

Copy-Number Variations in iExCN Genes Correlate with Survival Because our findings support the concept that multiple iExCN drivers can act within a single tumor, we tested whether the number of iExCN genes harboring CNVs influenced outcomes in children with FN RMS. We defined CNVs of iExCN genes as copynumber gain in any of the 25 oncogenes or loss in any of the 4 TSGs. Using two separate FN RMS cohorts, we found that cases with CNVs in %10 iExCN genes had significantly better failurefree and overall survival than those with >10 involved (log-rank test; p < 0.05 for each) (Figures 6A–6D). The finding was notable because the iExCN pipeline did not consider outcomes or clinical features such as tumor stage or clinical group, both of which correlate with outcomes for children with RMS (Malempati and Hawkins, 2012). The observation that children with a higher number of iExCN genes harboring copy-number changes had poorer survival supported the existence of gene-gene interactions among the iExCN genes. In that way, the effect of copy-number changes in a small number of iExCN genes may not be evident without the cooperative or synergistic effects of other genes. Three pieces of data indicated that poorer survival in cases with CNVs in a greater number of iExCN genes was not related merely to genomic instability. First, using an established chro-

The potential for next-generation sequencing to illuminate oncogenic drivers and deliver better therapy has yet to be fully realized for most cancers, including FN RMS. Working from the premise that gene expression changes drive the cancer phenotype, we developed a computational pipeline to identify gene expression changes encrypted by copy-number changes, anticipating them to be particularly important. This prediction is relevant for FN RMS because a 2015 DNA methylation analysis revealed that >12% of gene expression differences between FP and FN RMS are explained by DNA methylation (Sun et al., 2015). The iExCN analysis identified 29 FN RMS drivers and TSGs, most of which were not uncovered in previous genomics analyses (Chen et al., 2013; Paulson et al., 2011; Seki et al., 2015; Shern et al., 2014). While not discounting the importance of those approaches, we emphasize the value added by the complementary iExCN analysis. For example, the RAS/PIK3CA and cyclin D/CDK/RB pathways were previously implicated in RMS by SNVs and CDKN2A deletion (Chen et al., 2013; Shern et al., 2014). That iExCN identified PTEN, a negative regulator of RAS/PI3K signaling, and CCND2 and CDK6, negative regulators of RB, cements their importance in FN RMS and highlights coordinated copy-number and expression changes as mechanisms to corrupt those pathways.

DISCUSSION

Figure 4. Expression of iExCN Genes Falls with Skeletal Muscle Differentiation (A) Charts show gene expression for muscle differentiation markers (red), iExCN-derived oncogenic drivers (blue), and iExCN-derived TSGs (green) in human myoblasts cultured in growth medium (day 0) or differentiation medium for the indicated number of days (1–6). (B) Chart displays the number of iExCN genes (blue bars) or genes randomly chosen from all of those expressed in FN RMS (gray bars), with the indicated correlation to the degree of skeletal muscle differentiation. Pearson correlation coefficient near 0 indicates no correlation, whereas correlation coefficients of
1 or 1 indicate repression or induction of individual genes with muscle differentiation. (C) Charts display ENCODE ChIP-seq data of H3K4me1 and H3K9ac, both of which are histone markers of active promoters, and H3K27ac, which is a histone marker for active promoters and enhancers, in human myoblast and myotube on promoter regions of EZH2, PTN, SNAI2, or RIPK2. (D) Chart demonstrates that ectopically expressed MyoD augments the expression of a muscle creatine kinase (MCK) enhancer or promoter reporter in 10T1/2 fibroblasts, whereas co-transfection of EZH2, PTN, SNAI2, or RIPK2 blunts MyoD activity. Data are average values from replicate samples, normalized to a cotransfected Renilla luciferase. *p < 0.05 by two-tailed Student’s t test. (E) Charts show transient transfection of siRNA targeting EZH2, PTN, SNAI2, or RIPK2 or a scrambled control siRNA (+ or
, respectively) influences the expression of MYOG in the indicated fusion-positive (FP) and fusion-negative (FN) cell lines. *p < 0.05 by two-tailed Student’s t test. Data presented as mean ± SD from six replicates. See also Figures S4 and S5.

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A

B

C

D

E

F

G

H

I

Figure 5. siRNA and Pharmacological Inhibition of iExCN-Defined Oncogenes Decreases RMS Cell Accumulation (A–F) siRNA knockdown in three iExCN-predicted oncogenes (ZFHX4, SNAI2, and RIPK2) in three FN (JR1, RD, and RH18) and two FP (RH28 and RH30) cell lines. Charts display knockdown efficiency (A, C, and E) and inhibition of cell accumulation (B, D, and F) for siRNA knockdown. p values were calculated based on the two-tailed Student’s t test. KD, knockdown.

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Our analysis extends previous reports highlighting somatic SNVs (Chen et al., 2013; Seki et al., 2015; Shern et al., 2014) because by focusing on coordinate changes between gene expression and copy number, we identify previously unknown genes that contribute to FN RMS pathogenesis. This observation is supported by another report showing that other types of soft tissue sarcoma are also driven by CNV-coupled gene expression changes (The Cancer Genome Atlas, 2017). That iExCN genes are expressed and harbor CNVs across the majority of FN RMS cases also contrasts the narrow distribution of somatic SNVs, the majority of which we and others have shown to be found only in single FN RMS cases (Chen et al., 2013; Seki et al., 2015; Shern et al., 2014). In contrast, many individual FN RMS cases harbor CNVs in each of the iExCN genes, demonstrating their potential to represent commonly shared vulnerabilities. This was borne out in our CRISPR/Cas9 screen, showing vectors targeting two iExCN genes, EZH2 and SCARA3, to be depleted in both of the tested FN RMS lines. Both genes are expressed in >80% of RMS specimens and harbor copy-number gains in >50 of the approximately 200 cases that we analyzed. While EZH2 has been implicated in RMS and cancer (Ciarapica et al., 2014; Kim and Roberts, 2016), SCARA3 encodes a reactive oxygen species (ROS) scavenger receptor family protein with only limited connection to cancer. A previous genomics analysis concluded that genes controlling oxidative stress are important in RMS, but that analysis did not actually identify lesions in direct ROS regulators (Chen et al., 2013). SCARA3 represents one that was nominated in our study by iExCN and further highlighted in the CRISPR/ Cas9 mini-pool screen. Our findings help to explain mechanisms that underlie the wellknown terminal differentiation defect in RMS. Perhaps most important, finding that the expression of 14 iExCN-defined drivers falls with differentiation in normal skeletal myoblasts highlights the potential for multiple genes with low-level copynumber gains to contribute to a differentiation arrest in RMS. A number of those already are implicated in myogenic differentiation control. For example, D-type cyclins and CDK4/6 are recognized to impair normal muscle gene expression and contribute to RMS (Saab et al., 2011). Our candidates include CCND2 and CDK6, and we provide evidence that RIPK2, previously implicated as a regulator of myogenesis (Munz et al., 2002), contributes to muscle gene repression by influencing CDK4/6 activity. Aberrations in myogenic trans activators, such as MYOD1 mutation (Szuhai et al., 2014) and E2A splicing (Yang et al., 2009), align with our identification of EZH2 and SNAI2. The former encodes a histone methyltransferase in the polycomb repressor complex 2 (PRC2) that can help preserve a non-differentiated state of FN RMS (Ciarapica et al., 2014). SNAI2 encodes Slug, a zinc-finger transcription factor with a consensus DNA-binding element (CAGGTG), analogous to the CANNTG E-box bound by

MYOD-family proteins (Cobaleda et al., 2007; Saab et al., 2011). Proteins encoded by mouse Snai1 and Snai2 genes block MyoD1 binding and muscle differentiation in myoblasts (Soleimani et al., 2012). Further studies of the iExCN genes may reveal additional mechanisms by which they can impair either cell-cycle arrest or the activity of myogenic transcription factors, both of which are needed for terminal differentiation. Barriers to differentiation in RMS represent opportunities for therapeutics. In mammals, terminal skeletal muscle differentiation has long been known to impose an irreversible cell proliferation arrest (Saab et al., 2011). Even if complete differentiation cannot be achieved, higher expression of certain muscle genes portends better survival, which implies more favorable RMS biology (Davicioni et al., 2006). That multiple RMS drivers influence differentiation would explain why targeting an individual node (e.g., CDK4/6, which blocks differentiation in myoblasts) has limited impact (Saab et al., 2006; Skapek et al., 1995). However, the aforementioned coordinate repression of 14 iExCN genes provides a new entry point for ‘‘differentiation therapy.’’ The changes in histone methylation and acetylation at those genes may be choreographed in normal muscle by a small number of chromatin- or histone-modifying proteins, many of which already are targetable (Helin and Dhanak, 2013). While the increased number of low-level copy-number changes in iExCN genes drives higher expression of those genes and less favorable outcomes, we do not yet know how best to identify any single iExCN gene as a targetable vulnerability in an individual RMS specimen. As we show here, the propagation of two RMS lines is impaired by targeting individual genes, even though some do not harbor measurable copy-number changes. To help explain an apparent lack of specificity for iExCN prediction, we emphasize that iExCN represents a statistical tool that nominates disease genes by correlating gene expression and copy-number changes across many specimens. Obviously, the expression of any one of those genes in a cell line or individual tumor also may be driven by epigenetic mechanisms that are divorced from gene copy-number change. That also can explain how iExCN analysis of FN RMS can identify genes that are important in fusion-positive disease because the shared skeletal muscle lineage likely enables both RMS forms (or individual cancers within the same subtype) to co-opt ancestral ontogenetic themes to impair differentiation and drive cell proliferation, migration, and survival as targetable cancer hallmarks (Hanahan and Weinberg, 2011). The application of molecularly targeted therapies in childhood cancer is more likely to succeed if predictive biomarkers are identified, typically as a protein-altering mutation in a disease driver, as in the pediatric Molecular Analysis for Therapy Choice (MATCH) basket trial (Dolgin, 2017). Additional preclinical and clinical translational studies are needed to clarify how well gene copy number, expression, or both predicts any single iExCN gene to be a vulnerability in an individual tumor.

(G and H) Representative western blots (G) and qRT-PCR data (H) demonstrate that RIPK2 suppression by transient transfection of targeting (+) or scrambled (
) siRNA represses CDK4-dependent phosphorylation of RB and enhances expression of CDKN1A (p21Cip1) in the indicated fusion-positive (FP) and fusionnegative (FN) RMS lines. (I) Charts demonstrate that exposure of FP and FN RMS cells to RIPK2 inhibitors PP1 and ponatinib decreases RMS cell accumulation. *p < 0.05 by two-tailed Student’s t test. Data presented as mean ± SD from six replicates. See also Figures S3, S6, and S7.

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EXPERIMENTAL PROCEDURES RMS Cases and Genomic Datasets Genomic data from 290 specimens, collected from 290 patients and deidentified before use, were compiled from three sources referred to as the National Cancer Institute (NCI), the Children’s Oncology Group (COG), and the University of Texas Southwestern (UTSW) datasets (Table S1; Supplemental Experimental Procedures). Genomics analyses of archived patient samples were conducted at UTSW Medical Center with the approval of its institutional review board (STU 102011-034). Genomic Sequencing, Copy Number, and Gene Expression Data Analysis Details are provided in the Supplemental Experimental Procedures. In brief, whole-genome and WES reads were aligned to human reference genome (hg19), and somatic protein-altering mutations were identified by the Genome Analysis Tool Kit (GATK) pipeline. SNP arrays were processed by SNP-FASST segmentation algorithm implemented in Nexus BioDiscovery software (BioDiscoveryEl Segunda, CA). Significantly altered CNVs were examined by the GISTIC method using a default q value of 0.25 to define statistical significance, as previously described (Mermel et al., 2011). For gene expression data, RNA was extracted in the Triche lab and processed by the Affymetrix Exon 1.0 ST array platform according to the manufacturer’s recommendations (Affymetrix, CA). CEL files were analyzed using R/BioConductor with robust multiarray average (RMA) normalization and custom PERL scripts. Molecular Experiments Human myoblasts (Lonza) and RMS cell lines (JR1, RD, RH28, RH18, and RH30), either provided by P. Houghton (St. Jude Children’s Research Hospital) or obtained from the American Tissue Type Culture Collection, were cultivated, as previously described (Wilson et al., 2016). RMS cells were authenticated by RT-PCR for PAX3/FOXO1 fusion expression and by short tandem repeat (STR) testing to ensure distinct cell lines and compatibility with known standards (Figure S2A). Transient gene knockdown was achieved using siRNA duplexes (Life Technologies) and Lipofectamine RNAiMAX (Thermo Fisher Scientific). 4-Amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo-d-3,4-pyrimidine) (PP1) (10 mM) (EMD Biosciences) and ponatinib (Selleck Chemicals) were used to inactivate RIPK2. Expression plasmids encoding a subset of iExCN-defined drivers were obtained from GeneCopoeia or D.W. Abbott (Tigno-Aranjuez et al., 2010), with authentication by PCR or sequencing. MyoD activity was assessed using transient transfection and reporter assays, as previously described (Wilson et al., 2016). More experimental details are provided in the Supplemental Experimental Procedures. CRISPR/Cas9 Mini-pool Screen and Data Analysis A CRISPR/Cas9 lentiviral mini-pool screen was carried out in RD and JR1 cells (see Supplemental Experimental Procedures for full details). In brief, lentiviral vectors targeting iExCN-predicted genes and controls (Tables S5 and S6) were prepared and used as described (Shalem et al., 2014). Following transduction at low MOI, puromycin selection, and cultivation ex vivo for 14 days, genomic DNA was prepared for analysis in the UTSW McDermott Next Generation Sequencing Core using the Illumina NextSeq 500. The supporting reads were mapped to guide RNA sequences (Table S5) by a custom PERL script and then median normalized to adjust for the effect of library size and read count distribution. Average read counts for six guide RNAs per gene were used to represent gene-level lentiviral vector reads in each sample. The average gene-level read counts of three replicates from days 0 and 14 were

used to calculate fold change for each gene. Two-tailed Student’s t test with Benjamini-Hochberg FDR correction was used to adjust p values for multiple comparisons. Statistical Analyses Statistical analyses were conducted using R programming packages (https:// www.r-project.org/). The comparison of two independent samples was assessed using the two-tailed Student’s t test (normal distribution) or the nonparametric two-tailed Mann-Whitney U test (non-normal distribution). A p value <0.05 was considered statistically significant. For survival analysis (Figures 6A–6D), p values were calculated based on the log-rank test. Cohort I is based on published datasets (Paulson et al., 2011; Shern et al., 2014), while cohort II is from a unpublished dataset used in this study (COG dataset in Table S1). Note that study subject age and sex, available only on a subset of the data, were not incorporated into the survival analyses because those features are not generally accepted as influencing survival. Functional annotation of enriched gene sets was based on the Gene Ontology database (http://www. geneontology.org), and the hypergeometric test was used to determine statistically significant enrichment. p values were adjusted for multiple comparisons using the Benjamini-Hochberg procedure. Random sampling was performed using Statistics::R and List::Util modules in PERL and the ‘sample’ function in R to randomly choose elements from a population based on a user-defined number of elements and 10,000 iterations. DATA AND SOFTWARE AVAILABILITY The accession numbers for the genomic data reported in this paper are dbGaP: phs000720 and GEO: GSE114621. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, seven figures, and seven tables and can be found with this article online at https://doi.org/10.1016/j.celrep.2018.06.006. ACKNOWLEDGMENTS We are grateful to M. DePlaza, J. Singh, and other members of the Skapek laboratory for technical assistance and helpful comments, to P. Leavey (UTSW) for comments on the manuscript, and to C. Liu and J. Mendell (UTSW) for advice and early technical assistance with CRISPR/Cas9 targeting. We acknowledge support from the Hyundai Hope on Wheels Foundation, the Wipe Out Kids’ Cancer Foundation, the Children’s Cancer Fund, and the Patrick and Beatrice Haggerty Foundation, all of which were secured with assistance from the Children’s Health Children’s Medical Center Foundation, and from the Andrew McDonough B+ Foundation. Collection of expression and copy-number data in the COG dataset was supported by grants from the National Cancer Institute (U10CA098543 and U10CA098413). Work in the Skapek laboratory was also aided by grants from the Cancer Prevention and Research Institute of Texas (RP120685-P2) and the UTSW Harold C. Simmons Comprehensive Cancer Center from the National Cancer Institute (CA142543). AUTHOR CONTRIBUTIONS Conceptualization, J.F.A., L.X., S.X.S., Y.Z.; Software, L.X.; Bioinformatics and Statistical Analysis: L.X.; Investigation, D.R., J.L., S.S., Y.Z.; Resources,

Figure 6. Increased Number of iExCN Genes with CNVs Correlates with Survival in Two Independent FN RMS Cohorts (A–D) Kaplan-Meier plots display (A and B) failure-free and (C and D) overall survival in two independent cohorts with different numbers of iExCN genes with CNVs, as indicated. Cohorts I and II are described in the Supplemental Experimental Procedures. (E and F) Chart shows that the chromosomal instability index score in FN RMS cases with CNVs involving no more than 10 iExCN genes is not lower than in cases with CNVs in >10 iExCN genes, analyzed separately in cohort I (E) and II (F). (G and H) Chart shows that the average relative expression of 25 iExCN oncogenes is higher in FN RMS cases with CNVs in >10 iExCN genes in cohort I (G) and II (H). Data presented as mean ± SEM. See also Figure S7.

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D.S.H., D.R., J.K., J.F.S., S.X.S., T.J.T.; Data Curation, J.K., J.F.S., L.X., T.W.L., T.J.T.; Writing – Original Draft, L.X., S.X.S., Y.Z.; Writing – Review and Editing, J.F.A., D.S.H., D.R., J.L., L.X., S.S., S.X.S., T.W.L., T.J.T., Y.Z.; Supervision, S.X.S.; Funding Acquisition, J.F.A., S.X.S., Y.Z. DECLARATION OF INTERESTS The authors declare no competing interests. Received: December 6, 2017 Revised: March 29, 2018 Accepted: June 1, 2018 Published: July 3, 2018 REFERENCES Aguirre, A.J., Meyers, R.M., Weir, B.A., Vazquez, F., Zhang, C.Z., Ben-David, U., Cook, A., Ha, G., Harrington, W.F., Doshi, M.B., et al. (2016). Genomic copy number dictates a gene-independent cell response to CRISPR/Cas9 targeting. Cancer Discov. 6, 914–929. Andre´s, V., and Walsh, K. (1996). Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis. J. Cell Biol. 132, 657–666. Bain, J., Plater, L., Elliott, M., Shpiro, N., Hastie, C.J., McLauchlan, H., Klevernic, I., Arthur, J.S., Alessi, D.R., and Cohen, P. (2007). The selectivity of protein kinase inhibitors: a further update. Biochem. J. 408, 297–315. Canning, P., Ruan, Q., Schwerd, T., Hrdinka, M., Maki, J.L., Saleh, D., Suebsuwong, C., Ray, S., Brennan, P.E., Cuny, G.D., et al. (2015). Inflammatory signaling by NOD-RIPK2 is inhibited by clinically relevant type II kinase inhibitors. Chem. Biol. 22, 1174–1184. Cao, L., Yu, Y., Bilke, S., Walker, R.L., Mayeenuddin, L.H., Azorsa, D.O., Yang, F., Pineda, M., Helman, L.J., and Meltzer, P.S. (2010). Genome-wide identification of PAX3-FKHR binding sites in rhabdomyosarcoma reveals candidate target genes important for development and cancer. Cancer Res. 70, 6497– 6508. Chen, X., Stewart, E., Shelat, A.A., Qu, C., Bahrami, A., Hatley, M., Wu, G., Bradley, C., McEvoy, J., Pappo, A., et al.; St. Jude Children’s Research Hospital–Washington University Pediatric Cancer Genome Project (2013). Targeting oxidative stress in embryonal rhabdomyosarcoma. Cancer Cell 24, 710–724. Ciarapica, R., Carcarino, E., Adesso, L., De Salvo, M., Bracaglia, G., Leoncini, P.P., Dall’agnese, A., Verginelli, F., Milano, G.M., Boldrini, R., et al. (2014). Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS. BMC Cancer 14, 139. Cobaleda, C., Pe´rez-Caro, M., Vicente-Duen˜as, C., and Sa´nchez-Garcı´a, I. (2007). Function of the zinc-finger transcription factor SNAI2 in cancer and development. Annu. Rev. Genet. 41, 41–61. Davicioni, E., Finckenstein, F.G., Shahbazian, V., Buckley, J.D., Triche, T.J., and Anderson, M.J. (2006). Identification of a PAX-FKHR gene expression signature that defines molecular classes and determines the prognosis of alveolar rhabdomyosarcomas. Cancer Res. 66, 6936–6946.

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