Integrative Copy Number Analysis of Uveal Melanoma Reveals Novel

Published OnlineFirst June 21, 2019; DOI: 10.1158/1078-0432.CCR-18-3052

Translational Cancer Mechanisms and Therapy Clinical Cancer Research Integrative Copy Number Analysis of Uveal Melanoma Reveals Novel Candidate Genes Involved in Tumorigenesis Including a Tumor Suppressor Role for PHF10/BAF45a Hima Anbunathan1, Ruth Verstraten1,2, Arun D. Singh3, J. William Harbour4, and Anne M. Bowcock1,2,5

Abstract

Purpose: Uveal melanoma is a primary malignancy of the clusters of genes in focal copy number regions whose expres- eye with oncogenic mutations in GNAQ, GNA11, or CYSLTR2, sion was associated with metastasis and worse overall sur- and additional mutations in BAP1 (usually associated with vival. This included genes from Chr 1p36, 3p21, and 8q24.3. LOH of Chr 3), SF3B1,orEIF1AX. There are other character- At Chr 6q27, we identified two tumors with homozygous istic chromosomal alterations, but their significance is not deletion of PHF10/BAF45a and one with a frameshift muta- clear. tion with concomitant loss of the wild-type allele. Down- Experimental Design: To investigate genes driving chro- regulation of PHF10 in uveal melanoma cell lines and tumors mosomal alterations, we integrated copy number, transcrip- altered a number of biological pathways including develop- tome, and mutation data from three cohorts and followed up ment and adhesion. These findings provide support for a role key findings. for PHF10 as a novel tumor suppressor at Chr 6q27. Results: We observed significant enrichment of transcripts Conclusions: Integration of copy number, transcriptome, on chromosomes 1p, 3, 6, 8, and 16q and identified seven and mutation data revealed novel candidate genes playing a shared focal copy number alterations (FCNAs) on Chr 1p36, role in uveal melanoma pathogenesis and a potential tumor 2q37, 3, 6q25, 6q27, and 8q24. Integrated analyses revealed suppressor role for PHF10.

Introduction proﬁles (GEPs), some of which are predictive of metastatic risk (3, 4). Mutations in GNAQ, GNA11, CYSLTR2, and PLCB4 Uveal melanoma is the most common primary intraocular that constitutively activate Gaq signaling are seen in almost all malignant tumor diagnosed in approximately six cases per mil- tumors in a mutually exclusive manner (5–8). Inactivating muta- lion per year. Approximately 40% of patients develop metastatic tions in BAP1 at chromosome 3p21 (9) are found in tumors with melanoma to the liver within 10 years (1). Advances made in the loss of heterozygosity for Chr 3 (LOH3) and a GEP predictive of local methods of treatment of primary uveal melanoma have not metastatic risk (class 2 tumors; ref. 10), and mutations in SF3B1 or led to an improvement in survival and after metastasis there is a EIF1AX are found in tumors with disomy 3 and a GEP associated median survival time of less than 6 months (2). Uveal melanomas with an intermediate and low likelihood of metastasis, respec- display characteristic genomic signatures including recurrent tively (class 1 tumors; refs. 4, 11, 12). Whole-genome sequencing chromosomal aberrations, gene mutations, and gene expression has also revealed potential mutations in other genes (13). In addition to LOH3, uveal melanomas are characterized by chromosomal alterations that can include Chr 1p loss, 6p gain, 6q 1National Heart and Lung Institute, Imperial College, London, United Kingdom. loss, 8p loss, 8q gain, and 16q loss (14–20). These DNA copy 2Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, number aberrations (CNAs) are an important category of genetic New York, New York. 3Department of Ophthalmic Oncology, Cole Eye Institute, alterations that can lead to development and progression of 4 Cleveland Clinic, Cleveland, Ohio. Bascom Palmer Eye Institute, Sylvester cancers by affecting gene dosage, sometimes amplifying genes Comprehensive Cancer Center and Interdisciplinary Stem Cell Institute, Univer- conferring a proliferative or metastatic advantage or leading to sity of Miami Miller School of Medicine, Miami, Florida. 5Departments of Der- matology and Genetics & Genome Sciences, Icahn School of Medicine at Mount loss of function of tumor suppressor genes. However, the signif- Sinai, New York, New York. icance of CNAs in most cancers, including in uveal melanoma is poorly understood. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). A global characterization of copy number, transcriptome, mutation, and methylation status in uveal melanoma has been Corresponding Author: Anne M. Bowcock, Icahn School of Medicine at Mount described (20). This conﬁrmed much previous work and showed Sinai, One Gustave L. Levy Place, Box 1130, New York, NY 10029. Phone: 212-659- 8256; Fax: 212-987-2240; E-mail: [email protected] four distinct molecular subtypes of CNA, each associated with a varying degree of metastatic risk. Here, we describe copy number Clin Cancer Res 2019;XX:XX–XX analysis of 182 tumor samples from three different cohorts doi: 10.1158/1078-0432.CCR-18-3052 followed by integration of transcriptomic, methylation, and 2019 American Association for Cancer Research. mutation data to look for genes driving copy number loss or

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to an assay for chemotaxis as described elsewhere (27). Collagen Translational Relevance invasion (ECM552 Millipore) and adhesion assays (ECM545 Here we describe integration of copy number, transcrip- Millipore) were performed according to the manufacturer's tomic, epigenomic, and mutational data in uveal melanoma instructions. across three independent datasets. Although loss of one copy of chromosome 3 is usually accompanied by loss-of-function Western blot analysis mutations in BAP1, the significance of other chromosomal This was performed with standard approaches by querying a set changes such as loss of Chr 1p, 6q, and 8p and gain of Chr 6p of proteins identified following PHF10 knockdown in cell lines. and 8q are not clear. We describe candidate genes on altered The following antibodies were used: PHF10 (ab154637, Abcam), chromosomes affecting uveal melanoma pathogenesis and JAZF1 (ab80329, Abcam), SMAD2 (ab40855, Abcam), and patient survival. We also provide evidence for a tumor sup- PCGF3/5 (ab201510, Abcam). pressor role for PHF10 on Chr 6q27. Its loss affects early pathways in cell development such as transcriptional regula- Statistical analysis tion as well as adhesion and migration. This knowledge will be Three replicates per cell line were used for PHF10 knock- important as we progress toward a more comprehensive downs. Intergroup differences were assessed with a one-way molecular diagnosis of uveal melanoma and determination ANOVA with Bonferroni post hoc test. Results were expressed as of prognosis. Besides providing important insights in the mean and SD or in boxplots. The Kaplan–Meier method and development of uveal melanoma, these studies provide novel log-rank test were used to compare the survival plots and a therapeutic targets for this cancer. univariate Cox proportion hazard model was used to compare the effects of high and low expression levels of select genes on overall survival. Time was computed for days. Survival was defined as the elapsed interval until date of last follow-up or gain, extending earlier studies investigating the relationship with date of death. An effect was considered significant at an FDR chromosomal alterations and gene expression in uveal melanoma q-value less than 0.05. with microarrays (21). We describe candidate genes on chromosomes 1p, 8q, and 6q and provide evidence for a tumor suppressor role for PHF10 mapping to Chr 6q27. Functional studies provided Results insights into the consequences of loss of PHF10. Broad copy number alterations in uveal melanoma Genomic DNA samples from 182 primary enucleated uveal melanomas (87 with matched normal DNA) from three different Materials and Methods cohorts (Supplementary Table S2A) were profiled on three dif- Data source and analysis ferent high-resolution SNP platforms encompassing >700 K Data from 182 primary uveal melanoma tumor samples were probes with a median interprobe spacing of 24 to 18 Kb. The obtained from three different cohorts: Washington University analysis pipeline is described in Supplementary Fig. S1 and (WU), Cleveland Clinic (CC), and TCGA. All of the samples were revealed a high degree of nonrandom CNAs across samples from enucleated specimens obtained from adult patients after they had all cohorts. Twelve broad events affected >10% of all samples. This provided written informed consent. Studies were conducted in included Chr 1q gain, 6p gain, 8p gain, 8q gain, 21q gain and Chr accordance with recognized ethical guidelines (e.g., Declaration 1p loss, 3p loss, 3q loss, 6q loss, 8p loss, 9p loss, and 16q loss of Helsinki, CIOMS, Belmont Report, U.S. Common Rule) and (Supplementary Table S2B; Supplementary Fig. S2A). Chr 8q gain were approved by an institutional review board. The data analysis and LOH3 were detected in more than 50% of all samples, workflow and methods of analysis of SNP arrays (copy number), consistent with previous studies (15, 28). Ploidy estimates RNA sequencing (RNA-seq), and exome sequencing are described revealed an average of 2.4n in 103 tumors from the WU and CC in Supplementary Fig. S1. cohorts (Supplementary Fig. S2C) due to a minority of tumors being tetraploid that was more common in class 2 versus class 1 PHF10 knockdown and RNA sequencing tumors consistent with BAP1 deficiency being the prognostic Established cell lines derived from primary uveal melanomas predictor for patients with polyploid tumors (29). [Mel202 (22), 92-1 (23) and Mel290 (24) (described in Supple- Unsupervised hierarchical clustering of all uveal melanoma mentary Table S1)] were subjected to PHF10 knockdown with samples (N ¼ 182) revealed four distinct clusters similar to those siRNA (Santa Cruz Biotechnology, sc-95343) or a siRNA control described elsewhere (ref. 20; Supplementary Fig. S3): groups A that consisted of scrambled sequence/nonsilencing siRNA (Santa and B correspond to those classified as class 1A and 1B tumors, Cruz Biotechnology, sc-37007); siCtrl). RNA-seq was performed respectively, and groups C and D correspond to those classified as with routine methods. In the case of Mel202 and 92-1, RNA was class 2 tumors. Groups C and D were associated with monosomy sent to the Centre Nacional d'Analisi Genomica (CNAG, 3 and were highly significantly associated with metastatic out- Barcelona, Spain) for processing. In the case of Mel290 (25), come (P ¼ 1.44e08). The monosomy 3 tumors from both RNA was sent to Genewiz (www.genewiz.com). Raw counts were groups C and D showed gain of the entire 8q arm in nearly all analyzed with DeSeq2 (26). RNA-Seq data from cell lines are samples as reported previously in TCGA cohort (20). Patients available upon request. with monosomy 3, 8p loss, and 8q gain had lower overall survival (OS) as described elsewhere (30–35). When examined indepen- Migration, invasion, and adhesion assays dently, chromosomal alterations associated with metastatic dis- To assay for migration, cells were transfected in 6-cm dishes. ease were loss of 3p (P ¼ 2.29 10 8), loss of 3q (P ¼ 2.95 After 48 hours of incubation, they were trypsinized and subjected 10 7), loss of 6q (P ¼ 0.0246), gain of 8q (P ¼ 0.0001), and loss of

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Table 1A. Enrichment analysis between copy number and expression at the cytogenetic band level Cytogenetic band No. genes in gene set (K) TCGA k/K TCGA FDR P value CC k/K CC FDR q value Chr 16q22 168 0.3155 2.29 1031 ns ns Chr 1p34 195 0.3487 1.59 1043 ns ns Chr 1p35 116 0.3276 3.39 1023 ns ns Chr 1p36 504 0.244 5.51 1059 0.0496 6.97 1010 Chr 3p21 271 0.3542 8.61 1062 0.1218 2.08 1024 Chr 3p22 ns ns ns 0.1047 3.73 106 Chr 3p25 101 0.3564 2.29 1023 0.1584 1.58 1013 Chr 3q21 130 0.2615 2.36 1017 0.1308 4.22 1013 Chr 6p21 544 0.2059 6.75 1046 0.0551 4.52 1013 Chr 8p21 ns ns ns 0.0952 2.03 106 Chr 8p22 ns ns ns 0.186 2.37 107 Chr 8q13 ns ns ns 0.125 2.65 105 Chr 8q22 102 0.3137 6.55 1019 6.97 1010 Chr 8q24 214 0.3551 3.57 1049 0.1075 1.09 1015 NOTE: Results are presented for TCGA and CC cohorts where correlation coefﬁcientswere > 0.5. For each cytogenetic band the total number of genes analyzed, and the frequency of the signiﬁcantly correlated genes is shown, along with the P values and FDR P values. K is the number of genes in the set from MsigDB and k is the number of genes in the intersection of the query set with a set from MsigDB. The ratio of k/K in each cohort (CC and TCGA) is also provided.

8p (P ¼ 6.29 10 5). Chr 8p loss was more common in group C Focal copy number alterations in uveal melanoma and Chr 8p gain was more common in group D. We identified 77 The significance of most large-scale chromosomal changes in transcripts from Chr 8p that could explain this difference cancer is not known. Some are thought to reflect altered dosage of (Supplementary Fig. S4). a set of genes (e.g., loss of genes for lipid biosynthesis on Chr 8p in breast cancer; ref. 37). However, loss or gain of some chromosomes or chromosomal arms can reflect loss or gain of an Transcriptomic changes in regions with copy number alteration underlying tumor suppressor or oncogene, respectively, as with We integrated global copy number and transcriptomic data LOH3 and BAP1 (10). To search for genes driving regions of gain from TCGA and CC cohorts to identify transcripts driving the or loss in uveal melanoma, we looked for regions of common CNAs. We first performed a global correlation analysis between focal amplification or deletion and then asked which of the genes DNA copy number and transcript levels in the 80 tumor samples whose expression was correlated with CNA, resided in these from TCGA cohort and the 57 tumor samples from the CC regions. We then asked whether any of the genes in regions with cohort. Results are shown in Supplementary Table S3. There were focal alterations also harbored deleterious mutations in any uveal 922 transcripts whose expression levels were correlated with copy melanomas because this could be further evidence that the correct number changes at FDR P < 0.05 in both cohorts or within TCGA gene had been identified. alone where data for the CC cohort was missing (an additional Copy number data from the 182 uveal melanoma primary 1,218 transcripts). Testing for enrichment of genes by computing cancer specimens were analyzed with GISTIC (38). By employing the overlaps between our list and the Cytogenic set (C1) from a q value threshold of 0.1, 246 independent regions of signifi- MSigDB (36) revealed significant enrichment of genes/transcripts cantly recurrent somatic FCNAs were identified. These included on chromosomes 1p, 3, 6, 8, and 16q (Table 1A). Loss of 130 amplifications and 116 deletions. On average, after common transcripts from the cytoband in which BAP1 is located (3p21) germline variants were removed, there were 21 focal alterations was significant in both TCGA and CC cohorts and BAP1 showed per sample. Among these 246 regions, the CNA boundaries for strong correlation between copy number change and expression amplification events revealed a median size of 46.81 kb in both cohorts (correlation coefficient ¼ 0.77, FDR P ¼ 8 10 15 (0.6–7614 kb) and a median size of 259 kb (24–17328 kb) for and correlation coefficient ¼ 0.53, FDR P ¼ 0.001 in TCGA and deletions. For each of these significant FCNAs, a "peak" region CC, respectively). lying within a 99% confidence window was then identified that

Table 1B. High confidence overlapping GISTIC focal peaks in the of uveal melanoma cohorts, genes in minimal overlapping regions, and overlap with a Pan Cancer study Copy number GISTIC focal Overlapping Minimum overlapping Genes reported in event peak cohorts region (MOR) Genes in MOR Pan Cancer study (64) Amplification 8q22.1 WU & TCGA chr8:94721058-94749641 RBM12B, FAM92A1 Amplification 8q24.3 WU & CC chr8:144992,452- PLEC PARP10, CYC1 144993,067 Deletion 1p36.11 CC & TCGA chr1:26387424-26491354 PDIK1L, TRIM63, FAM110D SFN (lies nearby) Deletion 2q37.3 CC & TCGA chr2:241835544- SNED1, LOC200772, CROCC2, C2orf54 ING5 (lies nearby) 241943252 Deletion 3p11.1-q11.1 CC & WU Chr3:89516653-93735022 Centromere, EPHA3, PROS1, U3, ARL13B, STX19, U6 Deletion 6q25.2 CC & TCGA chr6:154470218- IPCEF1, CNKSR3, OPRM1 154726987 Deletion 6q27 WU & CC chr6:169652570- THBS2, WDR27, C6orf120, PHF10, TCTE3, 170152990 C6orf7

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Table 2. Copy number–driven gene clusters significantly associated with overall survival and metastasis in uveal melanoma Focal No. loci genes Genes associated with survival 8q24 13 COL22A1, ADCK5, ARC, C8orf33, CHRAC1, COMMD5, CYC1, DENND3, GPR20, MAFA, PTK2, PTP4A3, ZNF7 1p36 11 CNKSR1, EXTL1, NIPAL3, PAQR7, RCAN3, RHCE, UBXN11, WDTC1, HSPG2, PLA2G2C, IFFO2 3p21 9 BAP1, DNAH1, ITIH4, NISCH, SFMBT1, DUSP7, RPL29, PTPN23, SCAP 2q37 9 LIMS2, AGFG1, MFF, EIF4E2, GBX2, COPS8, HDAC4, HES6, ILKAP, KLHL30, PASK, SCLY 8q22 6 CDH17, FLJ46284, GEM, KIAA1429, PDP1, TMEM67 11p15 4 LRRC56, PNPLA2, RASSF7, SIRT3 6q23 4 RPS12, SLC2A12, HEBP2, NHSL1 6q24 4 CCDC28A, REPS1, EPM2A, SASH1 16q24 3 ZFPM1, MVD, ZNF778 2p22 3 DPY30, SLC30A6, SPAST 3p25 3 CAND2, RPL32, VGLL4 2q36 2 AGFG1, MFF 8q23 2 OXR1, ZFPM2 11q13 1 PC 14q21 1 LRR1 17p12 1 PMP22 19p13 1 PCSK4 1q25 1 TOR3A 20p13 1 SPEF1 20q13 1 ZNF512B 2p23 1 MEMO1 2q14 1 LIMS2 3q11 1 PROS1 3q28 1 CCDC50 4q24 1 CISD2 5p15 1 CCT5 6p21 1 FKBP5 6p22 1 RNF144B 6p25 1 SERPINB9 6q22 1 C6orf58 6q25 1 MTHFD1L 7q36 1 EZH2 8p22 1 MTUS1 Figure 1. 8q11 1 ATP6V1H Results of GISTIC analysis of copy number data from all three cohorts (TCGA, 8q13 1 SLCO5A1 WU, and CC). TCGA data included matched tumor/normal tissue for all 8q21 1 RUNX1T1 samples and germline CNVs could be filtered out in all cases. In the case of the WU and CC cohorts where matched normal DNA was not always available, germline CNAs were compiled from the Database of Genomic Variants (DGV) and HapMap normal and filtered out. Focal copy number further evidence that a biologically relevant interval has been alterations (FCNA) occurring at a significantly higher frequency in uveal identified. Globally, we identified seven regions with focal altera- melanomas compared with the average background rate are shown. tions that overlapped in two of the three cohorts (Table 2). Six of Overlapping peaks shared between any two cohorts are indicated. Red, these regions mapped to chromosomal regions with copy number gains; blue, losses. change already implicated in uveal melanoma: Chr 3, Chr 1p36, Chr 6q, and Chr 8q. These are discussed below. An overlapping region of deletion at Chr 1p36.11 was most likely to contain the putative locus involved in tumor- (chr1:26387424-26491354) deleted a segment of TRIM63, the igenesis. These peaks each contained a median of two genes entire PDIK1L gene and a segment of FAM110D. Of these genes, (range, 0–176) and included miRNAs and other noncoding TRIM63 showed the strongest correlation between deletion and RNAs). Thirteen regions contained more than 25 genes each, and expression in TCGA cohort (correlation coefficient ¼ 0.64, FDR the remaining 233 regions encompassed a total of 677 potential P ¼ 3.15 109; Supplementary Table S3). However, loss of target genes (Supplementary Table S4; Fig. 1). In addition, there expression of 1p is historically associated with poorer survival were 217 copy number and gene expression correlated tran- and some transcripts outside this region were strongly correlated scripts present within one or more reported GISTIC peaks across with this (see below). the three cohorts (Supplementary Table S3). Other regions of overlapping deletion were: Chr 2q37 (chr2:241835544-241943252) detected in 8% of tumors. This Overlapping focal changes in independent uveal melanoma region harbors SNED1, LOC200772, CROCC2, and C2orf54 but cohorts they did not exhibit correlation with between expression and Because focal alterations can be spurious and unrelated to copy number change. Chr 2q27 was also deleted in a Pan Cancer tumorigenesis, peaks from different cohorts that overlap provide study (ref. 38; ING5) and harbors HTR2B whose upregulation

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Figure 2. Region of homozygous deletion at Chr 6q27 in the CC and WU cohorts. The region of overlap that harbors PHF10, WDR27, and c6orf120 is shown. The location of the frameshift mutation identified in MM133 (a class 2 tumor) is also shown. The CC sample with the PHF10 HD was GSM1082739. The WU cohort sample with a PHF10 HD was MM016, a class 2 tumor in a female who was diagnosed with uveal melanoma in her 20s, did not harbor a detectable mutation in BAP1, SF3B1, or EIF1AX and who died from metastasis. discriminates class 2 from class 1 tumors (39). We also identified (c.678delT, p.F226fs; Supplementary Table S5) that was vali- an overlapping deletion in CC and WU cohorts around the Chr 3 dated with Sanger sequencing (Supplementary Fig. S5). Read centromere (chr3:89516653-93735022); and an overlapping counts revealed loss of the wild-type allele consistent with a deletion on Chr 6q25 (chr6:154470218-154726987) that har- potential role for PHF10 as a tumor suppressor. Tumor MM133 bors CNKSR3 (40). arose in a patient of 54 years of age. It was medium–large (19 At Chr 6q27, we identified an overlapping deletion of a 15 mm) of undifferentiated histology (epithelioid), no metas- region harboring WDR27, c6orf120, PHF10,andTCTE3 tasis at enucleation, and lacked a detectable BAP1 mutation. We (chr6:169652569-170152990). In the case of WDR27 and also identified a deleterious missense mutation (p.D453E, PHF10,expressionwascorrelatedwithdeletioninbothTCGA rs761295711) in PHF10 in TCGA tumor A8KM (Supplemen- and CC cohorts (Supplementary Table S3). Further inspection tary Table S5). of this region revealed that uveal melanoma patient MM016 There were two regions of copy number gain on Chr 8q. A (WU cohort) harbored a homozygous deletion of this region region at Chr 8q22.1 was detected in WU and TCGA cohorts (chr8: that resulted from a complex deletion of one Chr 6q homolog 94721058-94749641) and harbors RBM12B. The second region and a second deletion that spanned 54 kb at Chr6:170099399- mapped to Chr 8q24.3 (chr8:144992452-144993067) but only 170152990. This tumor had been diagnosed in a young woman harbored a segment of PLEC whose expression was only weakly of 24 years and it had very quickly metastasized. It had a correlated with copy number gain in TCGA (Supplementary Table differentiated histology (spindle), was very large (24 22 S3). However, a number of nearby transcripts from Chr 8q24.3 mm), and was disomic for Chr 3. It had the following chro- exhibited stronger correlation between copy number gain and mosomal changes: þ1q, þ6p, 6q, þ8, þ11p, 11q, þ22. expression and were also correlated with overall survival (dis- MM016 did not harbor a mutation in any previously identified cussed further below). prognostic driver (BAP1, EIF1AX, SF3B1) although it did harbor an oncogenic mutation in GNAQ and had upregulated PRAME Identification of additional candidate genes driven by (not shown) consistent with its metastatic features and its integrating copy number, exome sequencing, and CpG classification as a class IB tumor. Homozygous deletions are methylation rare and can point to the locale of tumor suppressors. Further Methylated CpG sites correlated with gene expression in both inspection revealed a second homozygous deletion spanning TCGA and CC cohorts are shown in Supplementary Table S6. this region (Chr 6:170073603-170123579; hg19) in a tumor of There were 1,984 transcripts with a significant correlation with a 91-year-old female patient from the CC cohort hypo- or hypermethylated CpG sites. Expression of 206 of these (GSM1082739). The overlapping regions of homozygous dele- transcripts was also correlated with chromosomal copy number tion in both of these tumors disrupted PHF10, C6orf120 and levels. These were primarily from Chr 3p21, 6p21, 3q21, 1p36, WDR27 (Fig. 2). Analysis of exome data also revealed a somatic 8p21, and 8q24 (Supplementary Fig. S6). Genes in deleted frameshift mutation in PHF10 in a class 2 tumor MM133 regions where there was also hypermethylation included EXTL1,

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RUNX3, and WASF2. These also resided in a region of focal system, and mRNA stability. The majority of these 1,072 tran- deletion at Chr 1p36.11 (chr1:18079813-27712917; Supplemen- scripts were from genes encoded by Chr 3p (165 genes), 3q tary Table S4). At several regions of copy number gain, there were (108 genes), 8q (152 genes), 6p (80 genes), and 1p (73 genes). clusters of upregulated genes exhibiting hypomethylation includ- Out of these 1,072 genes, 424 genes had a HR < 1 (lower ing on Chr 6p21.1 (KLHDC3, SLC29A1, BYSL, POLR1C, expression associated with good prognosis) and 647 genes had PPP2R5D, and XPO5) and Chr 8q24.3 (PTK2, PTP4A3, CYHR1, HR > 1 (lower gene expression associated with poor prognosis). PPP1R16A, RECQL4, and ZNF517). Upregulation of ENPP2, We focused on 92 genes present within focal regions of altera- mapping to 8q24.3 and previously reported as a gene expressed tions reported from our GISTIC analysis and compared the top at high levels in class 2 tumors (21) appears to have been driven three cluster of genes present at 8q24, 1p36, and 3p21 with exclusively by methylation (Supplementary Table S6). MTUS1, chromosomal alterations alone (Table 2). In the case of Chr 3, mapping to Chr 8p22 is one of the 12 prognostic transcripts (41) downregulation of BAP1 expression (HR ¼ 11.39; CI: 3.36–38.54; differentiating class 1 from the aggressive class 2 tumors. Its P ¼ 9.0e05) was more significant than monosomy 3 expression was correlated with both copy number and methyla- (HR ¼ 14.5; CI: 3.37–62.44; P ¼ 3.3e04) or the presence of tion in TCGA; however, it was not significant in the CC cohort BAP1 mutations (HR ¼ 6.58, CI: 2.4–17.89, P ¼ 2.18e04; (Supplementary Tables S3 and S6). Expression of MTUS1 was also Fig. 3A). In addition, there were eight other transcripts within significantly associated with metastasis and overall survival this cluster which were highly correlated with each other (Fig. 3A), (see below). two of which have been previously reported as downregulated in class 2 versus class 1 UM (RPL29 and SCAP; ref. 39). On Chr 1p36 Mutation analysis we identified a cluster of genes (Table 2) highly correlated with We looked for protein damaging mutations in genes affected poor outcome and associated with metastasis although 1p loss by copy number alteration (Supplementary Table S7). In addi- had not been significant in TCGA cohort (HR ¼ 1.62; CI: 0.66– tion to Chr 3p21, which harbors BAP1 and the handful of 3.98; P ¼ 0.29; Fig. 3B; Supplementary Table S3). A cluster of deleterious alterations in PHF10 at Chr 6q27, there was very transcripts at chromosome 8q24.3, encoded by 13 genes were little enrichment of mutations in any other single gene, and strongly correlated with metastasis and poor overall survival very few genes had the hallmarks of a tumor suppressor. On (Table 2; Fig. 3B). Compared with chromosomal 8q gain Chr 1p36, two tumors harbored mutations in MTOR,andat (HR ¼ 6.10; CI: 1.42–26.34; P ¼ 0.01) the HR were lower for Chr 1p34.1, one TCGA tumor (AA9A) harbored a mutation in the cluster of genes on 8q24 (Supplementary Table S3); however, KDM4A (exon13:c.C1942T:p.P648S) with loss of the wild-type the confidence interval are wide and more accurate estimates will allele. Exon 32 of PTK2 mapping to Chr 8q24.3 was somatically require larger sample sizes. MTUS1 on chromosome 8p was more mutated in MM127 (c.G3091T:p.A1031S), a class 2 tumor with strongly correlated with metastasis than Chr 8p gain (Supple- an activating mutation in GNAQ,noidentified mutation in mentary Table S3; Table 2; Fig. 3B), BAP1, SF3B1,orEIF1AX and no apparent copy number change (Supplementary Table S2B). CYC1 on Chr 8q24.3 was mutated Transcriptional profi ling of PHF10 knockdown in uveal in TCGA tumor A883 (p.D209delinsDYY). On Chr 16q23.1, melanoma cell lines MON1 homolog B (MON1B) showed the highest correlation We used siRNAs to investigate the consequences of PHF10 with expression in TCGA cohort (Supplementary Table S3) and knockdown in the established uveal melanoma cell lines was mutated in TCGA tumor A87Y (c.C1390T:p.R464X). A87Y Mel202, 92.1, and Mel290 (Fig. 3). We first profiled transcrip- was a primary tumor from an enucleated specimen that had tome-wide changes with RNA-Seq(SupplementaryTablesS9– metastasized. The same mutation has been described in an S11). All lines were wild type for PHF10 coding sequence, aggressive cutaneous neuroendocrine tumor (Merkel cell) although Mel202 and 92.1 had lost one copy of Chr 6q. There where Merkel cell polyomavirus has been implicated in a subset were 363 differentially expressed transcripts with a fold change of tumors (42). In rare cases, neuroendocrine tumors such as of 2 (adjusted P < 0.05) shared by at least two of the three cell those of Merkel cells can metastasize to the uvea (43). Hence, it lines (Fig. 4A). Significant gene ontology terms for these shared is possible that the uveal melanoma in A87Y represents a transcripts (Supplementary Table S12) included cell adhesion metastasis from a different primary tumor or that in some (GO:0007155), developmental process (GO:0032502), cell instances loss of function of MON1B is associated with the development (GO:0048468), regulation of multicellular development of uveal melanoma. organismal process (GO:0051239), and extracellular matrix organization (GO:0030198; Fig. 4B). Survival analysis Although there can be considerable differences in cell lines We asked how genes from regions exhibiting significantly high versus primary tumors, a number of transcripts altered by PHF10 correlation with expression and CNAs influenced patient overall knockdown exhibited the same trend in the PHF10-mutant survival and compared the results to chromosomal alterations tumors MM16 and A8KB (TCGA; Supplementary Table S13). alone. We performed a genome-wide univariate Cox proportional These included downregulated transcripts IL6ST, JAM3, JAZF1, hazard model and compared the effects of high and low gene PCGF5, SMAD2, and TRIM22 and upregulated transcripts PHRF1, expression in TCGA data. A total of 6,341 transcripts were signif- POLR3D, and TCF3. Many of these were transcription factors/ icantly associated with overall survival at q-value < 0.05 chromatin remodelers and Western blot analysis of the PHF10 (Supplementary Table S8). Next, we selected the most significant knockdowns with antibodies to some of the encoded peptides transcripts from regions of CNA and asked whether they were also confirmed altered protein expression of JAZF1, PCGF5, and associated with metastasis (Supplementary Table S3). This SMAD2 in the cell lines (Fig. 4C). resulted in 1,071 transcripts. Pathway enrichment analysis We also asked whether there were transcripts that were also revealed significant enrichment for metabolism of RNA, immune upregulated or downregulated in correlation with PHF10 changes

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Figure 3. Kaplan–Meier curves for overall survival (OS) in TCGA cohort (n ¼ 80) based on broad chromosomal changes versus candidate gene expression. In the case of BAP1, mutation status is included. A, Chromosome 3 loss versus BAP1 expression and mutation and a correlation plot showing the cluster of transcripts from chr 3p21 that are highly correlated with BAP1 expression. B, Examples for Chr 1p, 8q24.3 and 8p transcripts versus broad Chr 1p loss, 8q gain, and 8p loss. in the larger cohort of uveal melanomas. We categorized all 80 statistics. We then compared these two groups (PHF10 high and TCGA samples into those with low and high PHF10 gene expres- low) and identiﬁed 10,272 signiﬁcant differentially expressed sion based on the survival data using maximally selected rank transcripts (FDR P < 0.05; Supplementary Table S14A). There

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Figure 4. Consequence of PHF10 knockdown in three uveal melanoma cell lines (Mel202, 92.1 and Mel290). A, GO analysis of differentially expressed transcripts. For RNA sequencing, three biological replicates were prepared for each condition. Transcripts with a fold change >2 and adjusted P < 0.5 following PHF10 kd were selected, and those shared by at least two cell lines were used for pathway analysis with g:Profiler (https://biit.cs.ut.ee/gprofiler/gost). GO redundant terms were removed with REVIGO (65); B, Venn diagram showing the number of differentially expressed transcripts unique to and shared by each cell line; C, Western blot analysis of some transcripts encoding chromatin remodelers that were differentially expressed in PHF10-mutant tumors. Thirty micrograms of whole-cell lysate was loaded. Knockdown was confirmed with qRT-PCR, RNA-Seq, and Knockdown was confirmed with Western blotting and an antibody to PHF10 at a concentration of 1:1,000; D–F, Results of adhesion assays: 105 cells (PHF10kd or siCtrl for 24 hours) were seeded on top of a collagen insert in serum-free media and medium containing 10% FBS and extracellular matrix proteins were added to the bottom chamber. Cells were left to invade for 48 hours and then lysed and stained with CyQuant GR dye and fluorescence was measured at 480/520 nm. The extracellular matrix proteins tested here were Col I, Col II, and Col IV corresponding to Collagen 1, 2, and 4, respectively, FN (Fibronectin), LN (laminin), TN (tenascin), and VN (vitronectin). Neg corresponds to the negative control. Three biological replicates were analysed and results are shown with error bars represent SD of the mean (, P < 0.05). G, Result of migration assays performed as described elsewhere (27).

were 4,388 transcripts shared between TCGA PHF10 low samples significantly reduced migration in a chemotaxis assay in Mel202 and the UM PHF10 knockdown cell lines that included IL6ST, and 92.1, but not in Mel290 (Fig. 4G). JAM3, PCGF5, and PHRF1. Employing a more stringent filtering criteria for the TCGA data (FC > 2) there were 222 transcripts enriched in pathways including development (GO:2000026, Discussion regulation of angiogenesis (GO:0045765), and focal adhesion Here we describe large-scale and focal CNAs in uveal melanoma (KEGG:04510; Supplementary Table S14). identified by interrogating 182 primary tumors from three dif- We used migration, invasion, and adhesion assays to contrast ferent datasets and nominate candidate genes that may contribute cells treated with either a PHF10 suppressing siRNA or a non- to tumorigenesis following integration of gene expression, meth- silencing control. No change in invasion was observed with ylation and mutation data. Clustering of broad copy number PHF10 knockdown (Supplementary Fig. S8). However, PHF10 changes revealed four tumor groups characterized primarily by suppression trended toward less adhesion to most ECM proteins alterations involving 1p loss, 1p gain, monosomy 3, 6p gain, 6q in both Mel202 and 92.1 cell lines, but conversely showed no loss, 8p gain/loss 8q gain, and 16q loss. This is similar to what was effect in Mel290 (Fig. 4D–F). Knockdown of PHF10 resulted in recently described for TCGA (20) and previously identified with

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gene expression profiling alone (44). Although, there were no found in GNAQ in uveal melanoma activate YAP through FAK significant differences in the ploidy estimates between the class 1 and that inhibition of FAK reduces uveal melanoma growth (53). and class 2 subgroups, class 2 tumors tended to exhibit higher Chr 8q24 also harbors MYC, which is amplified in about 30% of ploidy states (tetraploidy), consistent with BAP1 deficiency being uveal melanomas (54), but its expression was not significantly the prognostic predictor for patients with polyploid tumors (29). upregulated as a consequence of 8q24 amplification. Integration of copy number with expression showed enrich- Integrated analyses also revealed that a cluster of genes on ment of genes in cytogenetic bands 1p36, 6p21, 8q22, 8q24, and chr1p36 is associated with metastasis and worse overall survival 3p21 (the locale of BAP1), consistent with comparative genomic compared with broad chromosomal changes. hybridization analysis (18). To search for candidate genes that At Chr 6q27, we obtained evidence for PHF10/BAF45a as a might be driving copy number alterations, we looked for focal novel tumor suppressor for uveal melanoma on the basis of three alterations and identified seven regions overlapping events in two tumors with definitive loss-of-function mutations: two tumors independent cohorts. Deletion events localized to chromosomes with overlapping homozygous deletion (HD) and one with a 1p36.11, 2q37, 3cen, 6q25, and 6q27 and amplification events frameshift mutation and concomitant loss of the wild-type allele mapped to 8q22.1 and 8q24.3. (MM133). GEP had classified one MM016 as a class 1B tumor and Loss of chromosome 3 was correlated with downregulation of MM133 as a class 2 tumor although they lacked SF3B1 and BAP1 expression of BAP1 (10). From our studies of clonality in uveal mutations respectively. However, in addition to the PHF10 frame- melanoma, BAP1 mutations were found to be occasionally pres- shift mutation, MM133 also harbored a mutation in SF3B1 ent in the subclones suggesting loss of one Chr 3 homolog leading to a R625C alteration and was disomic for Chr 3 despite precedes mutations in BAP1 (33). Thus, it is unlikely that hap- being classified as a class 2 tumor (Supplementary Table S2). We loinsufficienty of BAP1 is driving LOH3. However, we identified also identified a missense mutation in TCGA tumor A8KM an overlapping region of deletion at 3p11.1-q11.1 that contains (p.D453E, rs761295711). This mutation was very rare (frequency PROS1 and ARL13B as well as pericentromeric sequence. This has in Exac is 8.3 10 6) and although tumorigenicity of missense not been described before in the context of uveal melanoma and alterations is hard to predict, this alteration was predicted to be its significance is not clear and will require further investigation. damaging. Tumor A8KM had also lost one copy of Chr 3 (Sup- The region of 8q gain in uveal melanoma has been narrowed to plementary Table S1) and PRAME was upregulated but it lacked 8q23-q24 in a number of studies (16–19) and we identified focal identified mutations in BAP1, SF3B1,orEIF1AX (Supplementary amplification of a segment of PLEC at chr. 8q24.3. However, there Table S2). Hence, the presence of alterations in PHF10 and BAP1 were a number of candidates outside this region with higher were mutually exclusive in the small number of tumors sampled. correlation coefficients and significant P values (Supplementary Loss of 6q where PHF10 resides was not correlated with worse Table S3). 8q gainis associated with poor prognosis and expression overall survival in TCGA, nor were levels of PHF10. of a number of transcripts from Chr 8q24.3 exhibited high cor- PHF10 (AKA BAF45a) is a component of the PBAF complex (a relation with copy number gain. Their overexpression was more SWI/SNF-like complex) that is involved in chromatin remodel- significantly associated with metastasis and worse overall survival. ing (55), which in turn can affect transcriptional regulation. We also showed that in addition to Chr 8q24.3 gain, upregulation During PHF10 silencing, other components of the PBAF complex of PTK2 and PTP4A3 could be a consequence of demethylation. (SMARCE1, SMARCC1, and SMARCA4; Supplementary Fig. S7) These findings are consistent with earlier studies of PTP4A3 that were upregulated, potentially as a compensation mechanism. also showed it to be a strong predictor of metastasis where its RNA sequencing data were available for MM016 and A8KM and overexpression increased cell migration and invasiveness (45). we identified a number of chromatin remodelers with altered These results are also consistent with earlier array-CGH studies expression that exhibited the same trend in cell lines with PHF10 where the importance of chromosome 8q gain on metastasis was knockdown. We confirmed alteration at the protein level of some demonstrated, along with losses of 3, 8p, and 16q (46). of these including PCGF5, SMAD2, and JAZF1 (55). JAZF1 is PTK2/FAK is an established driver of some tumors and is a target required for ciliated cell differentiation in vitro (56) and is for amplification at 8q in primary hepatocellular carcinoma (HCC; involved in transcriptional repression. It can be fused to other ref. 47) and breast cancer (48). The number of copies of Chr 8q24.3 genes in endometrial stromal tumors where it disrupts the poly- in the region harboring PTK2 ranged from three to nine in the CC comb repressive complex 2 (PRC2), abolishes histone methyl and WU cohorts and given its association with poor overall transferase activity, and activates chromatin/genes normally survival, these data are consistent with earlier data, including a repressed by PRC2 (57). PCGF5 encodes a component of PRC1. clear association between metastatic potential of uveal melanoma It is required for the differentiation of mouse embryonic stem cells and 8q ploidy of five copies or more (13). Tumor MM127 (mESC) toward a neural cell fate where it functions both as a harbored an A1031S alteration in PTK2 and no other detectable repressor for the SMAD2/TGFb signaling pathway and as a facil- abnormalities. This alteration alters a highly conserved "DAKNL" itator for neural differentiation. Its loss impairs the reduction of motif in the C-terminal end of PTK2 that is conserved from H2AK119ub1 and H3K27me3 around neural-specific genes, humans to flies. It lies in a paxillin-binding subdomain of the keeping them repressed. FAT domain that is required for localizing PTK2 to focal adhesions PHF10 loss also led to downregulation of adhesion and cell in response to integrin stimulation (49, 50). We hypothesize that migration in Mel202 and 92.1 cell lines. This was not observed in this p.A1031S alteration strengthens cellular adhesion mediated Mel290, but this line lacks mutations in the known drivers BAP1, by FAK. Inhibition of FAK has been implicated in improving local SF3B1, EIF1AX so is not a "typical" uveal melanoma cell line. control in HPV-negative head and neck squamous cell carcinoma Moreover, this line is very migratory and not very adhesive at (HNSCC; ref. 51) and in castrate-resistant prostate cancer (52) and baseline. Some transcripts such as ITGA7, ITGA10, JAM3, LAMC1, might also be a therapeutic target in uveal melanoma. This is EPHB3, HES1, and PIK3CB that are involved in adhesion and consistent with recent studies showing that oncogenic mutations migration were downregulated after PHF10 kd. However some

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such as EPHA3, PPP1R9B, ADAM15, POSTN, and COL4A1 were Authors' Contributions upregulated, highlighting the complex relationship between Conception and design: H. Anbunathan, R. Verstraten, A.M. Bowcock metastasis, migration, and adhesion. Further studies will be Development of methodology: H. Anbunathan, R. Verstraten, A.M. Bowcock required to understand the role PHF10 plays in these processes. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.D. Singh, J.W. Harbour, A.M. Bowcock Mutations in PHF10 that drive tumor development have not Analysis and interpretation of data (e.g., statistical analysis, biostatistics, been described before, although mutations in other PBAF com- computational analysis): H. Anbunathan, R. Verstraten, A.M. Bowcock ponents such as ARID2, PBRM1, SMARCA4 (BRG), and SMARCB1 Writing, review, and/or revision of the manuscript: H. Anbunathan, (BAF47) are implicated in other cancer types (58, 59). PHF10 lies R. Verstraten, A.D. Singh, J.W. Harbour, A.M. Bowcock in a region of Chr 6q that harbors an unidentified tumor sup- Administrative, technical, or material support (i.e., reporting or organizing pressor for a variety of epithelial cancers (60–62), so it should also data, constructing databases): H. Anbunathan, A.M. Bowcock Study supervision: A.M. Bowcock be strongly considered as a candidate for this elusive gene (63). Acknowledgments Disclosure of Potential Conflicts of Interest This work was supported in part by NCI grant R01CA161870 (to A.M. Bowcock and J.W. Harbour) and R01CA125970 (to J.W. Harbour). We thank A.M. Bowcock is listed as a co-inventor on a patent entitled "Compositions Asif Chowdhury for technical assistance, Anita Rogic for help with the and Methods for Detecting Cancer Metastasis" to Washington University, migration assays, and Dr. Jacqueline Frost for editorial comments. We which is licensed to Castle Biosciences. A.D. Singh reports receiving speakers acknowledge core grant NCI No. CA1667 to MD Anderson that supports bureau honoraria from Eckert and Zeigler; holds ownership interest (includ- validation of cancer cell lines. ing patents) in Aura Biosciences; and is a consultant/advisory board member for Immunocore and Isoaid. J.W. Harbour is listed as inventor of a patent entitled "Method for predicting risk of metastasis" and coinventor on a patent The costs of publication of this article were defrayed in part by the payment of advertisement entitled "Compositions and Methods for Detecting Cancer Metastasis" page charges. This article must therefore be hereby marked in to Washington University, both licensed to Castle Biosciences, and is a accordance with 18 U.S.C. Section 1734 solely to indicate this fact. consultant/advisory board member for Castle Biosciences, Aura Biosciences, and Immunocore. No potential conflicts of interest were disclosed by the Received September 19, 2018; revised April 23, 2019; accepted June 17, 2019; other authors. published first June 27, 2019.

References 1. Aronow ME, Topham AK, Singh AD. Uveal melanoma: 5-year update on 14. Horsman DE, White VA. Cytogenetic analysis of uveal melanoma. Con- incidence, treatment, and survival (SEER 1973-2013). Ocul Oncol Pathol sistent occurrence of monosomy 3 and trisomy 8q. Cancer 1993;71:811–9. 2018;4:145–51. 15. Ghazvini S, Char DH, Kroll S, Waldman FM, Pinkel D. Comparative 2. Singh AD, Topham A. Survival rates with uveal melanoma in the United genomic hybridization analysis of archival formalin-fixed paraffin- States: 1973–1997. Ophthalmology 2003;110:962–5. embedded uveal melanomas. Cancer Genet Cytogenet 1996;90: 3. Worley LA, Onken MD, Person E, Robirds D, Branson J, Char DH, et al. 95–101. Transcriptomic versus chromosomal prognostic markers and clinical out- 16. Aalto Y, Eriksson L, Seregard S, Larsson O, Knuutila S. Concomitant loss of come in uveal melanoma. Clin Cancer Res 2007;13:1466–71. chromosome 3 and whole arm losses and gains of chromosome 1, 6, or 8 in 4. Decatur CL, Ong E, Garg N, Anbunathan H, Bowcock AM, Field MG, et al. metastasizing primary uveal melanoma. Invest Ophthalmol Vis Sci 2001; Driver mutations in uveal melanoma: associations with gene expression 42:313–7. profile and patient outcomes. JAMA Ophthalmol 2016;134:728–33. 17. Ehlers JP, Worley L, Onken MD, Harbour JW. DDEF1 is located in an 5. Van Raamsdonk CD, Bezrookove V, Green G, Bauer J, Gaugler L, O'Brien amplified region of chromosome 8q and is overexpressed in uveal mel- JM, et al. Frequent somatic mutations of GNAQ in uveal melanoma and anoma. Clin Cancer Res 2005;11:3609–13. blue naevi. Nature 2009;457:599–602. 18. Hughes S, Damato BE, Giddings I, Hiscott PS, Humphreys J, Houlston RS. 6. Van Raamsdonk CD, Griewank KG, Crosby MB, Garrido MC, Vemula S, Microarray comparative genomic hybridisation analysis of intraocular Wiesner T, et al. Mutations in GNA11 in uveal melanoma. N Engl J Med uveal melanomas identifies distinctive imbalances associated with loss of 2010;363:2191–9. chromosome 3. Br J Cancer 2005;93:1191–6. 7. Moore AR, Ceraudo E, Sher JJ, Guan Y, Shoushtari AN, Chang MT, et al. 19. Hammond DW, Al-Shammari NS, Danson S, Jacques R, Rennie IG, Sisley Recurrent activating mutations of G-protein-coupled receptor CYSLTR2 in K. High-resolution array CGH analysis identifies regional deletions and uveal melanoma. Nat Genet 2016;48:675–80. amplifications of chromosome 8 in uveal melanoma. Invest Ophthalmol 8. Johansson P, Aoude LG, Wadt K, Glasson WJ, Warrier SK, Hewitt AW, et al. Vis Sci 2015;56:3460–6. Deep sequencing of uveal melanoma identifies a recurrent mutation in 20. Robertson AG, Shih J, Yau C, Gibb EA, Oba J, Mungall KL, et al. Integrative PLCB4. Oncotarget 2016;7:4624–31. analysis identifies four molecular and clinical subsets in uveal melanoma. 9. Jensen DE, Proctor M, Marquis ST, Gardner HP, Ha SI, Chodosh LA, et al. Cancer Cell 2017;32:204–20. BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger 21. van Gils W, Lodder EM, Mensink HW, Kilic E, Naus NC, Bruggenwirth HT, and enhances BRCA1-mediated cell growth suppression. Oncogene 1998; et al. Gene expression profiling in uveal melanoma: two regions on 3p 16:1097–112. related to prognosis. Invest Ophthalmol Vis Sci 2008;49:4254–62. 10. Harbour JW, Onken MD, Roberson ED, Duan S, Cao L, Worley LA, et al. 22. Ksander BR, Rubsamen PE, Olsen KR, Cousins SW, Streilein JW. Studies of Frequent mutation of BAP1 in metastasizing uveal melanomas. Science tumor-infiltrating lymphocytes from a human choroidal melanoma. 2010;330:1410–3. Invest Ophthalmol Vis Sci 1991;32:3198–208. 11. Harbour JW, Roberson ED, Anbunathan H, Onken MD, Worley LA, Bow- 23. De Waard-Siebinga I, Blom DJ, Griffioen M, Schrier PI, Hoogendoorn E, cock AM. Recurrent mutations at codon 625 of the splicing factor SF3B1 in Beverstock G, et al. Establishment and characterization of an uveal- uveal melanoma. Nat Genet 2013;45:133–5. melanoma cell line. Int J Cancer 1995;62:155–61. 12. Martin M, Masshofer L, Temming P, Rahmann S, Metz C, Bornfeld N, et al. 24. Verbik DJ, Murray TG, Tran JM, Ksander BR. Melanomas that develop Exome sequencing identifies recurrent somatic mutations in EIF1AX and within the eye inhibit lymphocyte proliferation. Int J Cancer 1997;73: SF3B1 in uveal melanoma with disomy 3. Nat Genet 2013;45:933–6. 470–8. 13. Royer-Bertrand B, Torsello M, Rimoldi D, El Zaoui I, Cisarova K, 25. Hurks HM, Valter MM, Wilson L, Hilgert I, van den Elsen PJ, Jager MJ. Uveal Pescini-Gobert R, et al. Comprehensive genetic landscape of uveal mela- melanoma: no expresion of HLA-G. Inveset Ophthalmol Vis Sci 2001;42: noma by whole-genome sequencing. Am J Hum Genet 2016;99:1190–8. 3081–4.

OF10 Clin Cancer Res; 2019 Clinical Cancer Research

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26. Love MI, Huber W, Anders S. Moderated estimation of fold change 45. Laurent C, Valet F, Planque N, Silveri L, Maacha S, Anezo O, et al. High and dispersion for RNA-seq data with DESeq2. Genome Biol 2014; PTP4A3 phosphatase expression correlates with metastatic risk in uveal 15:550. melanoma patients. Cancer Res 2011;71:666–74. 27. Das TK, Dana D, Paroly SS, Perumal SK, Singh S, Jhun H, et al. Centrosomal 46. Trolet J, Hupe P, Huon I, Lebigot I, Decraene C, Delattre O, et al. Genomic kinase Nek2 cooperates with oncogenic pathways to promote metastasis. profiling and identification of high-risk uveal melanoma by array CGH Oncogenesis 2013;2:e69. analysis of primary tumors and liver metastases. Invest Ophthalmol Vis Sci 28. Horsman DE, Sroka H, Rootman J, White VA. Monosomy 3 and isochro- 2009;50:2572–80. mosome 8q in a uveal melanoma. Cancer Genet Cytogenet 1990;45: 47. Okamoto H, Yasui K, Zhao C, Arii S, Inazawa J. PTK2 and EIF3S3 genes may 249–53. be amplification targets at 8q23-q24 and are associated with large hepa- 29. Yavuzyigitoglu S, Mensink HW, Smit KN, Vaarwater J, Verdijk RM, Beverloo tocellular carcinomas. Hepatology 2003;38:1242–9. B, et al. Metastatic disease in polyploid uveal melanoma patients is 48. Naylor TL, Greshock J, Wang Y, Colligon T, Yu QC, Clemmer V, et al. High associated with BAP1 mutations. Invest Ophthalmol Vis Sci 2016;57: resolution genomic analysis of sporadic breast cancer using array-based 2232–9. comparative genomic hybridization. Breast Cancer Res 2005;7:R1186–98. 30. Damato B, Dopierala J, Klaasen A, van Dijk M, Sibbring J, Coupland SE. 49. Arold ST, Hoellerer MK, Noble ME. The structural basis of localization and Multiplex ligation-dependent probe amplification of uveal melanoma: signaling by the focal adhesion targeting domain. Structure 2002;10: correlation with metastatic death. Invest Ophthalmol Vis Sci 2009;50: 319–27. 3048–55. 50. Hayashi I, Vuori K, Liddington RC. The focal adhesion targeting (FAT) 31. Patrone S, Maric I, Rutigliani M, Lanza F, Puntoni M, Banelli B, et al. region of focal adhesion kinase is a four-helix bundle that binds paxillin. Prognostic value of chromosomal imbalances, gene mutations, and BAP1 Nat Struct Biol 2002;9:101–6. expression in uveal melanoma. Genes Chromosomes Cancer 2018;57: 51. Ribeiro IP, Marques F, Caramelo F, Pereira J, Patricio M, Prazeres H, et al. 387–400. Genetic gains and losses in oral squamous cell carcinoma: impact on 32. Dogrusoz M, Bagger M, van Duinen SG, Kroes WG, Ruivenkamp CA, clinical management. Cell Oncol (Dordr) 2014;37:29–39. Bohringer S, et al. The prognostic value of AJCC staging in uveal melanoma 52. Menon R, Deng M, Ruenauver K, Queisser A, Peifer M, Offermann A, et al. is enhanced by adding chromosome 3 and 8q status. Invest Ophthalmol Somatic copy number alterations by whole-exome sequencing implicates Vis Sci 2017;58:833–42. YWHAZ and PTK2 in castration-resistant prostate cancer. J Pathol 2013; 33. Field MG, Durante MA, Anbunathan H, Cai LZ, Decatur CL, Bowcock AM, 231:505–16. et al. Punctuated evolution of canonical genomic aberrations in uveal 53. Feng X, Arang N, Rigiracciolo DC, Lee JS, Yeerna H, Wang Z, et al. A melanoma. Nat Commun 2018;9:116. platform of synthetic lethal gene interaction networks reveals that the 34. Kivela TT, Piperno-Neumann S, Desjardins L, Schmittel A, Bechrakis N, GNAQ uveal melanoma oncogene controls the Hippo pathway through Midena E, et al. Validation of a prognostic staging for metastatic uveal FAK. Cancer Cell 2019;35:457–72. melanoma: a collaborative study of the European Ophthalmic Oncology 54. Parrella P, Caballero OL, Sidransky D, Merbs SL. Detection of c-myc Group. Am J Ophthalmol 2016;168:217–26. amplification in uveal melanoma by fluorescent in situ hybridization. 35. van Beek JG, Koopmans AE, Vaarwater J, de Rooi JJ, Paridaens D, Naus NC, Invest Ophthalmol Vis Sci 2001;42:1679–84. et al. The prognostic value of extraocular extension in relation to mono- 55. Krasteva V, Crabtree GR, Lessard JA. The BAF45a/PHF10 subunit of SWI/ somy 3 and gain of chromosome 8q in uveal melanoma. SNF-like chromatin remodeling complexes is essential for hematopoietic Invest Ophthalmol Vis Sci 2014;55:1284–91. stem cell maintenance. Exp Hematol 2017;48:58–71. 36. Liberzon A, Birger C, Thorvaldsdottir H, Ghandi M, Mesirov JP, Tamayo P. 56. Johnson JA, Watson JK, Nikolic MZ, Rawlins EL. Fank1 and Jazf1 promote The Molecular Signatures Database (MSigDB) hallmark gene set collection. multiciliated cell differentiation in the mouse airway epithelium. Biol Cell Syst 2015;1:417–25. Open 2018;7:1–11. 37. Cai Y, Crowther J, Pastor T, Abbasi Asbagh L, Baietti MF, De Troyer M, et al. 57. Ma X, Wang J, Wang J, Ma CX, Gao X, Patriub V, et al. The JAZF1-SUZ12 Loss of chromosome 8p governs tumor progression and drug response by fusion protein disrupts PRC2 complexes and impairs chromatin repression altering lipid metabolism. Cancer Cell 2016;29:751–66. during human endometrial stromal tumorogenesis. Oncotarget 2017;8: 38. Mermel CH, Schumacher SE, Hill B, Meyerson ML, Beroukhim R, Getz G. 4062–78. GISTIC2.0 facilitates sensitive and confident localization of the targets of 58. Kadoch C, Hargreaves DC, Hodges C, Elias L, Ho L, Ranish J, et al. Proteomic focal somatic copy-number alteration in human cancers. Genome Biol and bioinformatic analysis of mammalian SWI/SNF complexes identifies 2011;12:R41. extensive roles in human malignancy. Nat Genet 2013;45:592–601. 39. Onken MD, Worley LA, Ehlers JP, Harbour JW. Gene expression profiling in 59. Hodges C, Kirkland JG, Crabtree GR. The many roles of BAF (mSWI/SNF) uveal melanoma reveals two molecular classes and predicts metastatic and PBAF complexes in cancer. Cold Spring Harb Perspect Med 2016;6. death. Cancer Res 2004;64:7205–9. 60. Lin H, Morin PJ. A novel homozygous deletion at chromosomal band 6q27 40. Ziera T, Irlbacher H, Fromm A, Latouche C, Krug SM, Fromm M, et al. in an ovarian cancer cell line delineates the position of a putative tumor Cnksr3 is a direct mineralocorticoid receptor target gene and plays a key suppressor gene. Cancer Lett 2001;173:63–70. role in the regulation of the epithelial sodium channel. FASEB J 2009;23: 61. Saito S, Sirahama S, Matsushima M, Suzuki M, Sagae S, Kudo R, et al. 3936–46. Definition of a commonly deleted region in ovarian cancers to a 300-kb 41. Onken MD, Worley LA, Tuscan MD, Harbour JW. An accurate, clinically segment of chromosome 6q27. Cancer Res 1996;56:5586–9. feasible multi-gene expression assay for predicting metastasis in uveal 62. Morita R, Saito S, Ishikawa J, Ogawa O, Yoshida O, Yamakawa K, et al. melanoma. J Mol Diagn 2010;12:461–8. Common regions of deletion on chromosomes 5q, 6q, and 10q in renal cell 42. Harms PW, Vats P, Verhaegen ME, Robinson DR, Wu YM, Dhanasekaran carcinoma. Cancer Res 1991;51:5817–20. SM, et al. The distinctive mutational spectra of polyomavirus-negative 63. Aird D, Teng T, Huang CL, Pazolli E, Banka D, Cheung-Ong K, et al. merkel cell carcinoma. Cancer Res 2015;75:3720–7. Sensitivity to splicing modulation of BCL2 family genes defines cancer 43. Trichopoulos N, Augsburger JJ. Neuroendocrine tumours metastatic to the therapeutic strategies for splicing modulators. Nat Commun 2019;10:137. uvea: diagnosis by fine needle aspiration biopsy. Graefes Arch Clin Exp 64. Zack TI, Schumacher SE, Carter SL, Cherniack AD, Saksena G, Tabak B, et al. Ophthalmol 2006;244:524–8. Pan-cancer patterns of somatic copy number alteration. Nat Genet 2013; 44.LandrevilleS,AgapovaOA,HarbourJW.Emerginginsightsintothe 45:1134–40. molecular pathogenesis of uveal melanoma. Future Oncol 2008;4: 65. Supek F, Bosnjak M, Skunca N, Smuc T. REVIGO summarizes and visualizes 629–36. long lists of gene ontology terms. PLoS One 2011;6:e21800.

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Integrative Copy Number Analysis of Uveal Melanoma Reveals Novel Candidate Genes Involved in Tumorigenesis Including a Tumor Suppressor Role for PHF10/BAF45a

Hima Anbunathan, Ruth Verstraten, Arun D. Singh, et al.

Clin Cancer Res Published OnlineFirst June 21, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-3052

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