NRC 3087

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Advances in sarcoma genomics and new therapeutic targets

Barry S.Taylor*, Jordi Barretina||, Robert G.Maki, Cristina R.Antonescu*, Samuel Singer* and Marc Ladanyi*
Abstract | Increasingly, human mesenchymal malignancies are being classified by the abnormalities that drive their pathogenesis. Although many of these aberrations are highly prevalent within particular sarcoma subtypes, few are currently targeted therapeutically. Indeed, most subtypes of sarcoma are still treated with traditional therapeutic modalities, and in many cases sarcomas are resistant to adjuvant therapies. In this Review, we discuss the core molecular determinants of sarcomagenesis and emphasize the emerging genomic and functional genetic approaches that, coupled with novel therapeutic strategies, have the potential to transform the care of patients with sarcoma.
Translocations
Structural rearrangement that juxtaposes distant genome sequences, resulting in aberrant gene expression or modified regulatory control of a gene (promoter substitution) or the formation of a fusion gene that encodes an aberrant, chimeric protein (gene fusion). Pathognomonic translocations have more than diagnostic use, they are also the defining feature of the given tumour type.
*Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA. University of California, San Francisco, San Francisco, California 94143, USA. Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA. || Broad Institute, Cambridge, Massachusetts 02142, USA. Mount Sinai School of Medicine, New York, New York 10029, USA. Correspondence to M.L. e-mail: ladanyim@mskcc.org doi:10.1038/nrc3087 Published online 14 July 2011; corrected online 22 July 2011
Sarcomas are uncommon but diverse mesenchymal malignancies that arise in or from bone, cartilage or connective tissues, such as muscle, fat, peripheral nerves and fibrous or related tissues (FIG.1). Together, sarcomas affect ~11,000 individuals in the United States and ~200,000 individuals worldwide each year, they arise from multiple lineages, and they range from indolent to highly invasive and metastatic 1,2. From a molecular genetics perspective, they have traditionally been classified into two broad categories, each of which includes clinically diverse sarcomas. The first category comprises sarcomas with near-diploid karyotypes and simple genetic alterations, including translocations or specific activating mutations. The second category includes tumours with complex and unbalanced karyotypes. These tumours are typified by genome instability, which results in multiple genomic aberrations in the genome of a single tumour and heterogeneity of aberrations across tumours of a given type. The contrasting features of these two categories, which we first highlighted in 2002 (REF.3), have been well reviewed4. These categories are, however, broadly drawn and do not reflect the genetic diversity among tumours of a given type, the subtypes within classes or their diverse tumour biology (FIG.1). Most sarcomas with simple genetic alterations are translocation-associated sarcomas (which account for approximately one-third of all sarcomas). These tumours tend to arise denovo and, in some cases, harbour only a single defining cytogenetic abnormality that is present at initiation and is retained throughout their clonal evolution. The majority of gene fusions
resulting from these specific translocations encode chimeric transcription factors that cause the transcriptional dysregulation of target genes, whereas others encode chimeric protein tyrosine kinases or autocrine growth factors5. Although well studied, the physiological roles of the individual genes in these fusions have seldom been directly linked to their respective sarcoma phenotypes, except perhaps for translocations of the myogenic transcription factor genes paired box3 (PAX3) and PAX7 with forkhead box O1 (FOXO1) in alveolar rhabdomyosarcomas (ARMS) (discussed below)6. In contrast to translocation-associated sarcomas, some karyotypically complex sarcomas can arise from a less aggressive form and pass through discrete stages of progression that are accompanied by increasing genomic complexity. Examples include the progression from atypical lipoma or well-differentiated liposarcoma to dedifferentiated liposarcoma 79, the progression from neurofibroma to malignant peripheral nerve sheath tumour (MPNST)10,11 or the progression from enchondroma to chondrosarcoma 12. Importantly, however, most high-grade karyotypically complex sarcomas present denovo, without antecedent lowergrade lesions. A detailed listing of the genetic abnormalities in sarcomas and their conventional treatment is available elsewhere4,13,14. We focus here on the core mechanisms of pathogenesis in soft tissue sarcoma, the advanced genomic and functional genetic approaches being used for target discovery in this group of diseases and the novel therapeutic approaches for their treatment.
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At a glance
Human sarcomas are uncommon malignancies that arise from mesenchymal cell types, have varied genetic origins and are clinically heterogeneous. Many sarcomas arise denovo, driven by a single genetic abnormality, and some are progressive and harbour complexgenomes. Three core and context-dependent molecular mechanisms drive sarcomagenesis: dysregulation of gene expression by aberrant, chimeric transcription factors generated by specific gene fusions in translocation-associated sarcomas, somatic mutations affecting key signalling pathways and DNA copy-number abnormalities. Novel genomic findings from diverse approaches in sarcoma are identifying point mutations that co-occur with translocations, lineage-specific oncogenes, chromosome remodelling events, and both genomic alterations and mutations that alter canonical signalling and differentiation pathways. As integrative genomics and massively parallel sequencing increase the pace of discovery for the most common lesions in all but the rarest sarcomas, this necessitates renewed focus on developing invitro and invivo sarcoma models for accompanying target discovery and functional annotation of sarcoma genomes with genomics-guided functional genetics. Although conventional modalities predominate in sarcoma treatment, new approaches to target aberrant signalling with specific therapies, overcome acquired resistance and target unconventional pathways are rapidly evolving.
Myogenic
Originating in, or with expression specific to, muscular tissues.
Karyotypically complex
Tumours with a complex karyotype are those with numerical and structural abnormalities affecting multiple chromosomes in the nuclear genome.
Chromothripsis
A neologism coined to describe a proposed single catastrophic remodelling of a chromosome and its accompanying punctuated model of somatic cancer evolution.
Second-generation sequencing
Sequencing methods and associated chemistries that sequence >106 nucleic acid fragments in parallel, producing short reads of ~35400 bases. Used here synonymously with next-generation or massively parallel sequencing.
Molecular mechanisms of sarcomagenesis The mechanisms that drive human sarcomagenesis fall into three broad categories: transcriptional dysregulation owing to aberrant fusion proteins that result from genomic rearrangements (FIG.2a); somatic mutations in key genes and signalling pathways; and DNA copynumber abnormalities. The epigenetic underpinnings of sarcomagenesis are mostly still to be determined, at least insofar as data on recurrent methylation abnormalities in sarcoma genomes remain limited. Although this Review focuses on these three core oncogenic mechanisms and the distinct therapeutic modalities that may follow, some consideration of the pathogenetic mechanisms relating to chromosome translocations and genomic complexity or instability in sarcomas may be required. The perennial question of how and why translocations arise has been the subject of a recent review15. In sarcomas, as in many leukaemias, these seem to be fundamentally random events that become fixed through natural selection within the precursor cell. Insilico analysis of sequence and structure indicated that features such as overall gene size, average intron length and the length of the longest intron were all increased in translocation partner genes16. Additional factors increase the likelihood of random breaks in two genes leading to an illegitimate recombination event. These include the increased availability of translocation partner genes that is created by open chromatin conformation associated with gene transcription or replication, or the unexpected proximity of some partner genes owing to either the three-dimensional arrangement of chromosomes in the nucleus17 or coordinated transcription within the same transcriptional hubs. Importantly, so-called recombinogenic DNA sequence elements may be anecdotally involved18, but are not more frequent in translocated genes. More recently, the binding of a transcription factor, the androgen receptor (AR), has been implicated more directly in generating DNA strand breaks and consequent gene fusions1921, an observation so far restricted to
prostate cancer, but with intriguing implications for other hormone-driven cancers. Finally, in regard to external risk factors, sarcoma translocations in particular t(X;18) in synovial sarcoma may be rarely related to radiotherapy-induced DNA damage2224. Another aspect of genomic integrity is the mechanisms of telomere maintenance, of which two main types have been described in human tumours telomerase activation and the alternative lengthening of telomeres (ALT). These seem to differ in frequency between the major genomic classes of sarcomas. A predominance of telomerase activation in the absence of ALT seems to characterize sarcomas with specific chromosome translocations. Alternatively, ALT is frequently seen in sarcomas with nonspecific complex karyotypes25,26, and a connection between ALT and mesenchymal stem cell biology has been proposed27. Sarcomas with nonspecific complex karyotypes, but not translocation-associated sarcomas, are also occasionally seen in some hereditary syndromes that are associated with genomic instability, such as Werners syndrome (caused by mutations in WRN)28, Nijmegen breakage syndrome (caused by mutations in nibrin (NBN; also known as NBS1))29 and Rothmund Thomson syndrome (caused by mutations in RecQ protein-like 4 (RECQL4))30,31. Finally, a recent wholegenome sequencing study has found that some osteosarcomas and chordomas underwent a process termed chromothripsis. Rather than a multistep accumulation of unbalanced rearrangements, this is a single catastrophic genomic instability event that primarily affects a single chromosome32. Investigating the pathogenesis of chromothripsis and its occurrence in other sarcomas is of immediate interest.
Transcriptional target dysregulation Most translocation-associated sarcomas share a common biology of transcriptional target dysregulation. As noted above, most recurrent tumour type-specific translocations in sarcomas produce gene fusions that encode aberrant transcriptional proteins. The general biology of cancer gene fusions has been well reviewed33. Likewise, general reviews of translocation-associated sarcomas, including comprehensive listings of recurrent gene fusions in sarcomas, have recently been published5,14 (FIG.2a). Here, we limit the discussion to two aspects of transcriptional target gene dysregulation in translocation sarcomas that have been the focus of recent advances: first, the application of genome-wide transcription factor location analyses to comprehensively identify target genes of the fusion proteins, and second, the emerging evidence for aberrant nuclear reprogramming of mesenchymal stem cells in translocation-associated sarcomas. In the therapeutic avenues section below, we also discuss the use of the transcriptional targets of the fusion proteins as therapeutic targets, a third area of recent advances. Genome-wide approaches to define the target gene repertoires of sarcoma fusions have included chromatin immunoprecipitation (ChIP) coupled to arrays (ChIPchip), and more recently ChIP coupled to secondgeneration sequencing (ChIPseq) (BOX1). Both methods
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erans a arcoma protub ytom a Dermatobros m peric ngio c sarco tar) aema ti n s our-h blas pla se bro s tum ar/ ato brou lm ry m ato ary (pa bro Solit amm ses oin pe ato Myx -ty rom b oid ial sm rc ma a De pe Su elio m oth co a nd sar om ioe gio rc ng An is sa ma s hae po Ka
Adipocytic Chondro-osseous Fibroblastic or myobroblastic Fibrohistiocytic Nerve sheath Pericytic Skeletal muscle Smooth muscle Uncertain dierentiation Vascular
Re tifo Co rm mp ha osi te h ema Kap aem ngi osif ang oen orm d i hae man oend othe gioe oth lio ndo elio ma thel ma Malig iom nant myop a Malignant ericy glomus tu to mour (and variants) ma
Figure 1 | Taxonomy of soft tissue sarcoma. This unrooted phylogeny shows ~60 sarcoma subtypes, as originally defined by the World Health Organization International Agency for Research on Cancer1, amended and updated on the basis of current knowledge. The classification reflects relationships among lineage, prognosis (malignant, intermediate or Nature Reviews | Cancer locally aggressive, intermediate or rarely metastasizing), driver alterations and additional parameters. Branch lengths are determined by nearest neighbour joining of a discretized distance matrix based on the aforementioned variables. Initial branching reflects differences in lineage, with associated lineages appearing closer in distance (such as, skeletal and smooth muscle). Subsequent branching denotes similarity in prognosis, whether they are translocation-associated, and if so, the genes shared among distinct fusions (in this order). Although incomplete, as many subtypes lack sufficient global molecular profiling data on which to base a phylogeny, this initial formulation minimally reflects the relationships among lineage and major molecular lesions in the subtypes. The figure excludes 52 benign types of tumour. Note that the abbreviation MFH represents undifferentiated pleomorphic sarcoma; PNET, primitive neuroectodermal tumour.
identify binding sites for the aberrant fusion proteins but, unlike ChIPchip using commercial promoter arrays, ChIPseq is not limited to the regions that surround promoters. On integration with expression profiles, it is possible to determine whether the effect of a given fusion protein is predominantly repressing or activating. Mapping the genomic binding sites of the PAX3 FOXO1 fusion protein in ARMS cells has shown that binding is associated with the activation of transcription34. PAX3FOXO1 primarily binds to PAX3 sites outside the
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M es en ch ym al Ext ch ras on kel dr eta os l os teo arco sar m My com a xoi d/r a oun d-c Pleo ell mor lip phic lipo osarc sarc oma oma Dedierentiate d liposarcoma liposarcoma Well-dierentiated
Gastroin testina l strom al tu Deep-seated/visceral leiomyo mour sarcoma ma myosarco s leio Cutaneou arcoma os a abdomy oma rphic rh om rc Pleomo yosa rcomaa sarc om sa m r habd yo rco yo ing r ou dom yosa dom leros m Sc hab om tu b a ll r th bd rh -ce ha ea dle ar sh al r pin ol S on ve ve bry Al er Em ln ra he rip pe nt na g ali M
immediate vicinity of transcription start sites, typically >4 kilobases (kb) downstream. Co-enrichment of target PAX3 motifs with E-box motifs suggests the co-regulation of many target genes by other transcription factors that bind E-boxes34. The direct targets identified include myogenic genes, such as myogenic differentiation 1 (MYOD1) and myogenic factor 5 (MYF5), as well as many biologically interesting targets, such as fibroblast growth factor receptor 4 (FGFR4), anaplastic lymphoma receptor tyrosine kinase (ALK), MET, insulin-like growth factor1
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Adult brosarcoma Low-g Myxo rade bromy xoid sa Co Scle brosa rcoma rcom ros ng a ing en Gi epi ita an the l t-c lioi br d el os bro lt arc um sarc om om ou a a ro fs o tis su es
id elio ith Ep
r ou m tu tic ma as rco bl sa ro c sti b yo obla y m br or o r at my m e ou um am grad ct In owyti L ioc t his ro b H rm MF ma ifo FH elio ex -cell ory M Pl nt at pith FH yoe m Giaamm rphic M our/ m In omo tum ericu le ixed ma periph P M rdo ur oid tumo Cho bromyx Ossifying
Epithelioid sarcoma Extra Intim -renal rhabdoid tumour al s M Pe aligna arcoma riv nt m An asc gio ula esen ma chy re mo to pit ma id he b lio id ro ce us ll n his eo tio pla cy sm to ma
a om rc sa rt pa so oma c ar ol sar ve ial ma Al ov arco our Syn ar cell s -cell tum all round Cle lastic sm Desmop Ewings sa rcoma and PNET Extras keleta l my xoid ch ondro sarcom a
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PAX7FOXO1
TP S M4 SS S18 A 22 1 S LK 21 AS SS18 8SS SX2 PS S X4 20 CO C S L1A R1 X1 19 1 TFE TAF1 PDGF 3 5NR B 18 4A3 FUS 17 17 FUS CHOP CREB FUSCR 3L2 EB3L1 16 16 FUSATF1 TCF12NR4A3 15 15
EWSR1WT1 4A3 1NR EWSR R1FLI1 22 G EWS ER P SR1 EW CHO B1 E SR1 CR F1 EW SR1 1AT EW SR EW
12
TPM3 ALK
1 XO FO 3 X PA
14
14 13 12 11 10 7 9
9
13
Alveolar rhabdomyosarcoma Alveolar so -part sarcoma Angiomatoid brous histiocytoma Clear-cell sarcoma Congenital brosarcoma Dermatobrosarcoma protuberans Desmoplastic round-cell tumour Endometrial stromal sarcoma Ewings sarcoma Inammatory myobroblastic tumour Low-grade bromyxoid sarcoma Extraskeletal myxoid chondrosarcoma Myxoid/round-cell liposarcoma Pericytoma Synovial sarcoma
6
11
b
80
85
90 q
K3 TR N V6 ET
8
8
LI1 G F1 TB PH 12 AC ZF1 SUZ JA F1 JAZ
30
EPC1 PHF 1
75
35 14 13
15
16
17 18
19 20 21 22
X 1 2 3
70
chr12 40 11 10 9 65
12
4 8 7 6 5
45
Intermediate-complexity dedierentiated liposarcoma
60 55
50
Figure 2 | The structure of sarcoma genomes. a | A summary of the recurrent translocations in malignant soft-tissue sarcomas indicates shared fusion partners between subtypes and regions of the genome that are subject to more frequent Nature Reviews | Cancer rearrangement. The outer ring represents genomic location (as labelled), cytobands are shaded and the centromere is in red, with curves joining fusion partners. b | The right panel shows the somatic structure of an intermediate-genomically complex sarcoma, a dedifferentiated liposarcoma (whole genome), as defined by long-insert low depth-of-coverage mate-paired second-generation sequencing (BOX1) (B.S.T. and S.S., unpublished data). Intrachromosomal rearrangements are shown in orange and interchromosomal rearrangements in dark red; a subset of the interchromosomal rearrangements is reminiscent of some recurrent translocations involving chromosome 12 (chr12) in part a. The left panel shows the pathognomonic chromosome 12q amplification in greater detail. This detailed view indicates a dense network of back-and-forth inverted and non-inverted intrachromosomal rearrangements in three clusters (in grey, purple and blue; for clarity, interchromosomal rearrangements are excluded). The curves reflect rearrangements between two genomic loci, as determined experimentally and computationally. ACTB, actin-; ALK, anaplastic lymphoma receptor tyrosine kinase; ASPSCR1, alveolar soft-part sarcoma chromosome region candidate 1 (also known as ASPL); ATF1, activating transcription factor 1; CHOP, C/EBP-homologous protein (also known as DDIT3); COL1A1, collagen typeI1; CREB1, cAMP responsive element binding protein 1; CREB3L, cAMP responsive element binding protein 3-like; EPC1, enhancer of polycomb homologue 1; ETV6, ets variant 6; EWSR1, Ewing sarcoma breakpoint region 1 (which encodes EWS); FLI1, Friend leukaemia virus integration 1; FOXO1, forkhead box O1; FUS, fused in sarcoma; JAZF1, JAZF zinc finger 1; NR4A3, nuclear receptor subfamily 4A3; NTRK3, neurotrophic tyrosine kinase receptor 3; PAX, paired box; PDGFB, platelet-derived growth factor-; PHF1, PHD finger protein 1; SUZ12, suppressor of zeste 12; TAF15, 68 kDa RNA polymerase II TATA box binding protein-associated factor; TCF12, transcription factor 12; TFE3, transcription factor binding to IGHM enhancer 3; TPM, tropomyosin; WT1, Wilms tumour 1.
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Box 1 | Advances in cancer genome and transcriptome characterization
Second-generation sequencing is enabling nucleotide-resolution oncogenomics188. Paired-end (or mate-paired) sequencing involves sequencing of short stretches of DNA on both ends of a larger fragment and aligning to the reference genome. Atypically aligned pairs (those with an unexpected position, orientation or separation distance) often reflect genomic rearrangements such as translocations (FIG.2b). Paired-end sequencing is therefore a powerful method for ascertaining structural rearrangements and marks the first time that this information has been readily available in an unbiased manner. It is suitable for the detection of rearrangements from variable depth-of-coverage whole-genome sequencing, and its sensitivity increases as the fragment length increases. These methods will help to detect previously unknown driver fusions in sarcomas with highly complex karyotypes. Paired-end sequencing can also be used in RNA sequencing of tumour transcriptomes, as has been done for prostate cancers and other malignancies189,190. To explore the biology of chimeric transcriptional proteins in translocation-associated sarcomas, chromatin immunoprecipitation coupled to sequencing (ChIPseq) can determine fusion protein location, facilitating target gene discovery. Finally, deep whole-exome and whole-genome sequencing, described in detail elsewhere188, have a central place in sarcoma genomics. Exome sequencing first captures and then deeply sequences all protein-coding exons of human genes. By contrast, whole-genome sequencing is unbiased, sequencing all accessible regions in the human genome. Both experiments detect point mutations and small insertions and deletions (indels) in exons, whereas whole-genome sequencing can simultaneously capture genome structure (in paired-end format; described above) and crucially, intergenic variation. Intergenic germline variation or somatic mutations, although currently under-explored, could have an important role in sarcomagenesis. This is typified by the MDM2SNP309 promoter polymorphism191, which, along with MDM2 amplification and TP53 deletion and mutation, represents another (albeit more subtle) mechanism of p53 pathway alteration in a broad range of sarcomas.
distant regions together in a transcriptional hub to allow EWSFLI1 to modulate gene expression. As microsatellites are known polymorphic sites, it has been hypothesized that higher repeat content at one or more key target genes may underlie individual or ethnic differences in Ewings sarcoma susceptibility for example, its rarity in individuals of African descent41. EWSFLI1 also binds to more conventional, nonrepetitive ETS motifs, and these sites are associated with genes that show either repression or activation of transcription42. A subset of EWSFLI1 target regions shows co-enrichment of sites for other transcription factors: E2F, nuclear respiratory factor 1 (NRF1) and subunits of nuclear transcription factor Y (NFY), raising the possibility of specific cooperative interactions43. In general, the combination of genome-wide target gene identification with gene expression data should accelerate the discovery of genes that are crucial to tumour growth and survival in translocation sarcomas. Genes that are found to be directly upregulated by specific aberrant sarcoma fusion proteins can be subjected to focused RNA interference (RNAi)-based screens to identify the genes that are most essential to the sarcoma in question (see Target discovery section below). Reprogramming. Recent efforts to generate nonembryonic stem cells have renewed interest in nuclear or lineage reprogramming 44,45. Understanding reprogramming may also inform our ideas of translocation sarcomas that are driven by aberrant transcription factors. Assigning lineage to translocation sarcomas has proved difficult, as is the case for Ewings sarcoma, synovial sarcoma, ASPS and others. The cell-of-origin for each of these has long been debated, especially owing to another peculiar clinical feature of these sarcoma types: their occurrence in unusual sites for tumours of bone and soft tissue, such as the kidneys, lungs or pancreas. One explanation for both characteristics is an origin from more than one stem or progenitor cell type or from related precursor cells in different parts of the body, with the similar undifferentiated or aberrantly differentiated phenotypes resulting from nuclear reprogramming by the aberrant transcription factors. For example, it has been shown that EWSFLI1, the fusion that defines Ewings sarcoma, can induce neuroectodermal gene expression in heterologous cell types, such as fibroblasts and rhabdomyosarcoma cells46,47. Indeed, this scenario has previously been postulated48, and is supported by compelling data from recent studies, although some disagreements remain49. Silencing EWSFLI1 in Ewings sarcoma cell lines produces an expression profile that is most similar to mesenchymal stem cells (MSCs) or mesenchymal progenitor cells50,51, and these can be subsequently induced to differentiate along adipogenic or osteoblastic lineages51. Thus, EWSFLI1 induces a limited neuroectodermal gene expression programme and imposes a differentiation block on MSCs (or a related cell type52), including a block on osteogenic differentiation by inhibiting runt-related transcription factor 2 (RUNX2) binding to genes that are associated with osteogenic differentiation53. In the converse experiment,
Paired-end (or mate-paired) sequencing

A technique whereby a library of genomic DNA or double-stranded cDNA is created and circularized, and then short stretches (35400 bp) are sequenced from either end of the cleaved product, but not the intervening variable-length fragment (from 200500 bp to 310 kb).
RNA interference
A technique for sequence-specific gene silencing in which small non-coding RNAs (principally microRNAs and small interfering RNAs (siRNAs)) and associated regulatory complexes pair with complementary mRNA targets.
Neuroectodermal
Of the neuroectoderm, including neural crest and neural tube cell types.
receptor (IGF1R) and MYCN, in some cases confirming previous single-gene studies3537. The role of some of these PAX3FOXO1 target genes in sarcomagenesis is discussed furtherbelow. In alveolar soft part sarcoma (ASPS), the ASPS locus (ASPL; also known as ASPSCR1) gene fuses with the transcription factor binding to IGHM enhancer 3 (TFE3) gene to form a chimeric protein that retains the TFE3 DNA-binding domain and therefore its CACGTG recognition site. In ChIPchip studies, we have found that ASPLTFE3 localization is predictably enriched at this canonical site and is exclusively associated with the activation of target genes, including MET38, cytochrome P450 17A1 (CYP17A1) and uridine phosphorylase 1 (UPP1)39. A somewhat more complicated picture has emerged for the major Ewings sarcoma fusion, which involves Ewing sarcoma breakpoint region 1 (EWSR1, which encodes EWS) and Friend leukaemia virus integration1 (FLI1). Several ChIP data sets have been generated in different Ewings sarcoma cell lines with endogenous EWSR1FLI1 fusion, all using the same FLI1 antibody for immunoprecipitation of EWSFLI1-bound DNA. The numbers of bound genomic regions in such studies have varied widely 4042. ChIPseq subsequently demonstrated that most of the genomic regions that were bound by EWSFLI1 were intergenic and that, through the ETS family DNA-binding domain (from FLI1), EWSFLI1 binds strongly to GGAA microsatellites40,41. Microsatellites containing six or more GGAA repeats (the core ETS domain-binding sequence) are associated with EWSFLI1 target gene upregulation40,42. These repeats are often more than 200 kb upstream of the target gene transcription start site, suggesting that chromatin looping brings
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EWSFLI1 expression in human MSCs induces a Ewings sarcoma-associated gene expression profile, which is especially clear in MSCs that are derived from younger individuals54,55. By contrast, EWSFLI1 expression in differentiated cell types with an intact ARFp53 pathway induces apoptosis or growth arrest 46. In human MSCs, EWSFLI1 directly upregulates the Polycomb group transcriptional repressor enhancer of zeste homologue 2 (EZH2)56 and induces expression of embryonic stem cell genes POU5F1 (also known as OCT4), SRY-box 2 (SOX2) and NANOG, at least partly by repressing mir145 expression54. Interestingly, EWSR1 also fuses with POU5F1 itself, albeit rarely, in undifferentiated bone sarcoma57,58, myoepithelial tumours of the soft tissue59 and certain salivary gland tumours60. Synovial sarcomas contain fusions of SS18 (also known as SYT) with either SSX1 or SSX2 (this fusion is referred to as SYTSSX below). In a striking analogy to the EWSFLI1 data, synovial sarcoma cell lines also express POU5F1, SOX2 and NANOG, and silencing of SYTSSX in these cell lines enhances their potential to differentiate along adipogenic, osteoblastic or chondrogenic lineages61. The formation of synovial sarcomalike tumours in mice with conditional expression of SYTSSX2 in myoblasts 62 or other lineages63 can be interpreted as further evidence of nuclear reprogramming by the fusion protein in a variety of more-or-less committed mesenchymal lineages. Finally, the sarcoma fusions of myxoid liposarcoma (fused in sarcoma
Table 1 | Recent genomic discoveries in sarcoma
Subtype Gene Alteration
Point mutations Overexpression Point mutations
(FUS)C/EBP-homologous protein (CHOP; also known as DDIT3)) and ARMS (PAX3FOXO1) have also been reported to transform mouse mesenchymal stem or progenitor cells64,65.
Mutations in key genes and signalling pathways Excluding the gene fusions in translocation sarcomas, few highly recurrent driver genes have been described in sarcoma. The major exception is gastrointestinal stromal tumour (GIST). GIST, one of the more common types of human sarcoma, is characterized by oncogenic mutations in KIT, less often in platelet-derived growth factor receptor- (PDGFRA) and rarely in BRAF6668. In fact, the dependence of GIST on constitutively activated KIT and PDGFR has led to treatment with selective kinase inhibitors (discussed below), representing a paradigm of targeted therapy in solid tumours. Oncogenic mutations occur in several different domains of KIT, and the location affects sensitivity to targeted inhibitors. Levels of KIT are also high in interstitial cells of Cajal, the presumed cell of origin for GIST. Nevertheless, oncogenic KIT mutations in the activation domain (D816V in particular) are also found in tumours of diverse lineages, including mastocytosis, acute myeloid leukaemia and germ celltumours. Approximately 10% of adult GISTs lack a KIT or a PDGFRA mutation, and a small subset (<1% of total GIST cases) harbour BRAFV600E mutations66 (TABLE1). In total, approximately 10% of adult and most paediatric
Frequency Discovery platform

1820% ~100% ~1%
Comment
Refs
74 196 66 32
Myxoid/round-cell PIK3CA liposarcoma GIST GIST Osteosarcoma or chordoma Dedifferentiated liposarcoma Leiomyosarcoma ETV1 BRAF Multiple
Sanger sequencing and Helical and kinase domain mutations seem mass-spectrometric genotyping to be biologically and clinically distinct Combined informatics and biochemical characterization Hotspot genotyping Paired-end DNA sequencing Cooperates with KIT mutation to mediate transformation Mutually exclusive with KIT and PDGFRA mutations Rearrangements occur in a catastrophic chromosome event, termed chromothripsis Amplification and overexpression of JUN represses C/EBP function, blocking adipocyte differentiation Retroperitoneal leiomyosarcoma differentiation dependent on MYOCD gain of function Distinguishes epithelioid haemangioendothelioma from morphological mimics A defining and diagnostic gene fusion in these sarcomas Heterozygous losses, homozygous deletions and inactivating mutations in complex sarcomas Central and periosteal tumours only
Structural ~25% rearrangements Amplification ~25%
JUN
Microsatellite loci and custom BACPAC aCGH BACPAC aCGH and expression analysis Positional cloning by FISH
9,91
MYOCD
Amplification
~70%
100, 101 197
Epithelioid haemangioendothelioma Mesenchymal chondrosarcoma
WWTR1 CAMTA1 HEY1 NCOA2
Fusion gene
~100%
Fusion gene
~100%
Exon array profiling SNP arrays and Sanger sequencing Mass-spectrometric genotyping
198 74
Myxofibrosarcoma NF1 and pleomorphic liposarcoma Chondrosarcoma or chondroma IDH1 or IDH2
Deletions, point 811% mutations and indels Point mutations ~56%
199
aCGH, array comparative genomic hybridization; BACPAC, bacterial artificial chromosomeP1-derived artificial chromosome; CAMTA1, calmodulin binding transcription factor 1; CEBP, CCAAT/enhancer binding protein-; ETV1, ets variant 1; FISH, fluorescence insitu hybridization; GIST, gastrointestinal stromal tumour; HEY1, hairy/ enhancer-of-split related with YRPW motif 1; IDH, isocitrate dehydrogenase; indel, insertion/deletion; MYOCD, myocardin; NCOA2, nuclear receptor co-activator 2; NF1, neurofibromin 1; PDGFRA, platelet-derived growth factor receptor-; SNP, single nucleotide polymorphism; WWTR1, WW domain containing transcription regulator 1.
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GISTs harbour no mutations in KIT, PDGFRA or BRAF, although KIT pathway activity is high. Among these, paediatric tumours show consistent overexpression of IGF1R mRNA and protein, although the mechanism remains unknown, as no genomic amplifications or activating mutations have been described at the IGF1R locus. In fact, paediatric tumours have mostly diploid genomes with few, if any, DNA copy-number alterations 69. Therefore, ongoing deep sequencing in paediatric GISTs is being used to identify alternative oncogenic events. A particularly attractive method may be hybrid capture of protein-coding exons followed by second-generation sequencing (exome sequencing (BOX1)). Although generally sporadic, GISTs can also present as part of syndromes such as familial GIST, Carneys triad, CarneyStratakis syndrome and neurofibromatosis typeI. In CarneyStratakis syndrome, which is characterized by the co-occurrence of GIST and paraganglioma, germline mutations in genes encoding subunits of succinate dehydrogenase (SDH) have been identified, as is also the case in familial paraganglioma70. Most GISTs that occur in association with neurofibromatosis typeI harbour somatic inactivation of the wild-type neurofibromin 1 (NF1) allele, whereas very few have KIT or PDGFRA mutations71,72. A role for tyrosine kinases is also emerging in angiosarcoma, a highly aggressive vascular tumour, in which transcription profiles show striking overexpression of vascular-specific receptor tyrosine kinases, including kinase insert domain receptor (KDR; which encodes vascular endothelial growth factor receptor 2 (VEGFR2)), TIE1, SNF-related kinase (SNRK), TEK and fms-related tyrosine kinase 1 (FLT1)73. Sequencing of these five genes has revealed KDR mutations in about 10% of angiosarcoma cases. The VEGFR2mutant proteins, when expressed in COS-7 cells, showed ligand-independent activation73. A recent large-scale analysis of the genomic landscape of sarcomas that encompassed seven major subtypes (myxoid/round-cell liposarcoma; dedifferentiated liposarcoma; pleomorphic liposarcoma; myxofibrosarcoma; leiomyosarcoma; GIST; and synovial sarcoma) identified frequent mutations in TP53 (which encodes p53), NF1 and PI3K catalytic subunit- (PIK3CA)74. TP53 mutations were identified in 17% of pleomorphic liposarcomas, which is consistent with these mutations occurring frequently in sarcomas with complex karyotypes75,76. By contrast, in translocation-associated sarcomas, secondary genetic alterations, such as TP53 mutations or homozygous deletions of cyclin-dependent kinase inhibitor 2A (CDKN2A), are less common but, when present, are associated with a highly aggressive clinical course77. The discovery of PIK3CA mutations in 18% of myxoid/round-cell liposarcomas (TABLE1) raised the possibility that secondary mutations may cooperate with the FUSCHOP fusion protein in oncogenesis74. Mutations in PIK3CA clustered in the same two hot spots that are observed in epithelial tumours the helical domain (E542K and E545K) and the kinase domain (H1047L and H1047R). Patients with helical domain mutations had a shorter disease-specific survival and increased phosphorylation of AKT at both CREB-regulated transcription coactivator 2 (CRTC2; also known as TORC2) and pyruvate dehydrogenase kinase1 (PDK1) phosphorylation sites than patients with wild-type or kinase domain-mutant tumours74. Another novel finding is the identification of NF1 alterations (point mutations or deletions) in 10% of myxofibrosarcomas and 8% of pleomorphic liposarcomas74 (TABLE1). NF1 germline and somatic mutations are typically associated with NF1 inactivation in sarcomas in individuals with neurofibromatosis typeI, but NF1 mutations had not previously been described in sporadic sarcomas.
Carneys triad
A rare syndrome in which there is a coexistence of three distinct tumour types: GIST, extra-adrenal paraganglioma and pulmonary chondromas.
CarneyStratakis syndrome
Distinct from Carneys triad, and also referred to as the GIST-paraganglioma dyad, a heritable syndrome in which familial mutations are associated with coexisting GIST and paraganglioma, but not pulmonary chondromas.
Neurofibromatosis typeI
An autosomal dominant genetic disorder in which tumours arise from nerve tissues and from all neural crest cell types.
Paraganglioma
An uncommon neuroendocrine tumour arising from the sympathetic component of the autonomic nervous system and found predominantly in the abdomen, chest or head and neck region.
Disease-specific survival
Patients with a given diagnosis who do not die of the specified disease in a defined period of time, which excludes patients who died from causes other than the studied disease.
Genomic copy-number alterations DNA copy-number alterations are the third core mechanism of sarcomagenesis. Sarcomas span a wide range of complexity among human malignancies in terms of their copy-number alterations78. They vary from translocationassociated sarcomas with generally few copy-number alterations, either broad or focal, to karyotypically complex subtypes that are heterogeneous, unstable and profoundly altered in genomic copy number. In addition, a recent high-resolution array-based copy-number analysis revealed a category of sarcomas with intermediate complexity that were mainly characterized by few, but highly recurrent amplifications, as exemplified by dedifferentiated liposarcomas74. These and similar genomic data support an alternative sarcoma classification to the one based on low-resolution karyotypes. These three groups are genomically simple sarcomas that are driven by pathognomonic translocations or point mutations, non-translocation-associated sarcomas of intermediate genomic complexity; and highly genomically complex sarcomas. However, some subtypes may not fit so neatly into these broad groups, such as PAX7FOXO1-positive ARMS. Data from another copy-number analysis show that the third category can be subdivided into sarcomas with few chromosome arm or whole-chromosome gains or losses and sarcoma genomes with a high level of chromosome complexity 79. The first group, genomically simple sarcomas, harbour characteristic gene fusions or activating mutations that are thought to represent early events in their pathogenesis. However, even these tumours can acquire genomic complexity in advanced stages of disease80,81. Intermediate complexity sarcomas are exemplified by well-differentiated and dedifferentiated liposarcomas, which are mainly driven by chromosome 12 amplifications, often manifesting as double minute chromosomes, ring chromosomes and larger markers82 (FIG.2b). These 12q gains have high prevalence (8090%), and the coamplified oncogenes cyclin-dependent kinase 4 (CDK4) and MDM2 can serve as confirmatory diagnostic markers83 and as potential pharmacological targets74,84,85. The structure, stability and reintegration of these amplicons into liposarcoma genomes can also alter their affect on oncogenic phenotypes86. Another gene that is affected by 12q amplification is high mobility group AT-hook2 (HMGA2), which often loses its 3 untranslated region (UTR), disrupting microRNA-mediated repression87. This genomic remodelling of chromosome 12 is
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probably the result of progressive rearrangement and amplification in an evolving amplicon rather than of a single catastrophic event, such as the recently proposed chromothripsis that is seen in a subset of osteosarcomas and chordomas32 (TABLE1). Similar 12q amplifications occur at lower frequencies in other mesenchymal tumours, such as osteosarcomas88, as well as several epithelial tumourtypes78. Other notable, albeit less recurrent, amplifications in intermediate-complexity sarcomas occur on 1p and 6q. These amplifications, which seem to be mutually exclusive, span genes in the p38 and JNK pathways of MAPK signalling including, JUN (TABLE1) on 1p, and TAK1-binding protein 2 (TAB2) and MAP3K5 (also known as ASK1; a kinase upstream of JUN) on 6q9,8991. An additional target of genomic amplification is telomerase reverse transcriptase (TERT) on 5p74. Some targets of genomic amplification seem to be shared among a subset of both intermediate and highly complex sarcomas, including Yes-associated protein 1 (YAP1) and vestigial-like 3 (VGLL3) on 11q22 and 3p12, respectively 92. Finally, highly complex sarcomas harbour multiple numerical and structural chromosome aberrations that are reminiscent of the vast majority of epithelial tumours. The molecular classification of these subtypes reflects the varying levels of similarity in their genomic aberrations; some subtypes may be considered as a single entity 93, while others are distinct 94. Broad amplifications of several chromosome arms (such as 5p95) often occur in combination with deletions that affect well-established tumour suppressors such as CDKN2A, CDKN2B, PTEN, retinoblastoma 1 (RB1), NF1 and TP53. The affected gene, if not homozygously deleted, often harbours an inactivating mutation in the remaining allele74. In fact, several of the products of these genes have a direct role in maintaining chromosome integrity 96,97, and their loss of function may be an early event that leads to genomic instability in highly complex sarcomas. In other subtypes, such as leiomyosarcoma, genomic deletions are more common than amplifications74,98. Nevertheless, at least a subset of leiomyosarcomas depends on the specific amplification of myocardin (MYOCD), which encodes a smooth muscle-specific transcriptional co-activator of the serum response factor (SRF)99101 (TABLE1). The involvement of MYOCD in smooth muscle differentiation implies that it may serve as a lineage-survival oncogene102. Therefore, although systematic catalogues of copy-number alterations point to pathways that are potentially activated in specific subtypes, to precisely delineate the genes that are involved in the events that drive sarcomagenesis it will be essential to annotate genomic characterization with high-throughput functional genetics for target discovery. technologies, which detect not only genome-wide copy-number changes, but also rearrangements and mutations (BOX1). Thus far, large genomic characterization efforts in cancer have mainly focused on epithelial and haematological cancers. Given the need for new therapies for sarcomas, their inclusion in such studies is expected in the near future. For example, The Cancer Genome Atlas (TCGA) project (see Further information) is initiating a comprehensive genomic analysis of dedifferentiated liposarcoma, leiomyosarcoma and undifferentiated pleomorphic sarcoma, although this will have to overcome a perennial challenge in sarcoma genomic research the scarcity of samples. Nevertheless, given the large number of differentiation lineages among diverse sarcomas, a detailed genetic characterization of these tumours is likely to benefit our wider understanding of cancer ingeneral. Genomics-guided functional genetics. A gene that is recurrently altered in a sarcoma subtype does not necessarily have a role in cancer initiation or progression. In fact, the identification of recurrent lesions (BOX2) far outstrips our ability to test their importance. To determine the involvement of a gene in sarcoma biology and to assign it as a therapeutic target, systematic biological validation in genetically defined models must follow. Furthermore, even when a causal role for a given genetic alteration is experimentally supported in a particular cancer type, the crucial downstream targets may remain elusive and so require further functional studies. However, functional studies in sarcoma are hampered by the dearth of such appropriate models. Only limited numbers of human sarcoma cell lines exist, in part because of the rarity of certain diagnoses and the resulting scarcity of samples. Moreover, for each of the subtypes with complex genomes, multiple cell lines are needed to represent the diversity of genetic alterations within that subtype. Several large-scale projects now aim to genetically characterize large numbers of human cancer cell lines and screen these against a range of anticancer therapies to correlate drug sensitivity with genetic markers. Among these are the Cancer Cell Line Encyclopedia (J.B., personal communication) and the Sanger Cancer Cell Line Project 103, which is assembling approximately 800 cell lines, of which only ten (1.3%) represent complex soft-tissue sarcomas (another 38 represent Ewings sarcoma or primitive neuroectodermal tumours, rhabdomyosarcoma or osteosarcoma). There is, therefore, a pressing need to generate cell lines that are representative of diverse sarcoma types, mainly for the subtypes with complex karyotypes. The creation of a sarcoma cell line panel with cytogenetic and genomic profiles that mirror the diversity that is observed in their corresponding tumour types would represent an important step in dissecting the influence of heterogeneity on variability of response to targeted therapies104. Such a panel could also drive genomics-guided functional genetics, either with arrayed or pooled loss-of-function RNAi screens105,106, or with gain-of-function ORFeome approaches107 (FIG.3).
www.nature.com/reviews/cancer 2011 Macmillan Publishers Limited. All rights reserved
Response
Used here as defined by response evaluation criteria in solid tumours (RECIST) criteria. The RECIST criteria are guidelines developed to document a change in tumour burden and similarly monitor response to treatment during the clinical evaluation of cancer therapeutics.
ORFeome
A collection of cloned human protein-coding open reading frames (ORFs) suitable for stable expression via destination vectors in model systems (see Further information).
Target discovery Systematic surveys of cancer genomes with integrated genomics have proved to be an effective approach for identifying targetable genetic alterations in specific cancer types. The list of potential targets is expected to grow with the expanded use of second-generation sequencing
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Box 2 | Identifying candidate driver alterations in noisy cancer genomes
Over the course of their somatic evolution, cancer genomes can acquire an array of abnormalities. These alterations either confer a clonal growth advantage to the cell (driver) or are acquired stochastically, but are biologically neutral (passenger). The need to distinguish between these two alteration types in increasingly complex genomic data has driven the development of robust and statistically principled computational methodologies. Alteration type-specific methods have particularly focused on DNA copy-number alterations (CNAs), one of the most common somatic genetic events not only in karyotypically complex sarcomas, but also in the genomes of epithelial cancers. Two such methods, Genomic Identification of Significant Targets in Cancer (GISTIC)192 and RAE193, not only assign a significance to candidate driver alterations emerging from a background of random, passenger abnormalities using their pattern of recurrence, amplitude and extent, but also assign to individual tumours the set of CNAs they have undergone. The outputs of these computational methods can be used in studies of clinical associations, analyses of aberrant pathway activity, integrated with orthogonal data, or used to populate large-scale functional genetic screens. Alternatively, other methods, such as iCluster194 and Copy Number and Expression in Cancer (CONEXIC)195 identify putative driver alterations by integrating multiple high-throughput data types (such as, expression and copy number).
Along these lines, we recently sought to functionally annotate the dedifferentiated liposarcoma genome by systematically knocking down genes that are altered by recurrent genomic amplification on 12q and elsewhere. With an arrayed loss-of-function short hairpin RNA (shRNA) screen, we determined which amplified genes are required for cell proliferation and survival74. We concentrated on dedifferentiated liposarcomas because the marked homogeneity of their genetic alterations compensates for the low number of cell lines available. Profiling of three dedifferentiated liposarcoma cell lines showed that this small panel captured a substantial number of the molecular abnormalities that are observed in primary tumours. Using these validated cell lines, we identified several genes that are required for cancer cell viability, some of them potentially druggable. For example, the hits included not only CDK4 at 12q14, confirming its importance in this sarcoma, but also aurora kinase A (AURKA) at 20q13, specific inhibitors of which are currently in clinical trials108. These studies also provided a setting in which we could address an open question in cancer genetics namely, whether focal genomic amplifications contain a single driver gene or, as recently suggested, multiple independent drivers109. We found evidence that MDM2 and YEATS4, which are frequently co-amplified with each other (and nearly always in the same tumours with CDK4 amplification) are both drivers74. MDM2 is a validated target in this disease, as drugs that inhibit the MDM2p53 interaction induce apoptosis in dedifferentiated liposarcoma cell lines84,85. Therefore, these data support the concept of multiple driver genes in a single amplicon and hint at a more complex effect of genomic amplification on cancer phenotypes than has previously been understood. Furthermore, co-amplified genes may influence phenotypes that are unrelated to viability, so alternative assays are needed to test their role as oncogenes. Overall, this study design establishes a framework for the systematic genomic and functional genetic characterization of other rarecancers.
Invivo models of sarcoma. In addition to cell lines, several other types of models have been used for sarcoma and are probably similarly adaptable to invivo functional genetics for target discovery. These include exvivo cultures of tissue slices that preserve the original tumour microenvironment 110 and low-passage shortterm cultures111,112, both of which are tractable surrogates of primary tumours. Nevertheless, to test novel targeted therapies, it is essential to develop invivo models of sarcomas. Both subcutaneous and orthotopic xenografts (injecting sarcoma cell lines into immunocompromised mice) have been used to model human sarcomas, but these model systems also have certain limitations. Some genetic abnormalities that are present in primary tumours will not be present or retained in the xenografts, and, conversely, serial passaging can introduce additional alterations that do not reflect the primary tumours. Indeed, many treatments that initially showed promise in these models have not successfully translated to the clinic. To overcome these limitations, researchers are attempting to create panels of xenografts directly from primary tumour tissues that represent several sarcoma subtypes113. An alternative to xenografts is to genetically engineer animal models that reproduce the characteristics of human tumours, but this presents specific challenges. For mouse models of translocation-associated sarcomas, the challenge is to express the fusion oncogene in the correct lineage and stage of development. For mouse models of karyotypically complex sarcomas, the challenge is to express genuine alterations in an appropriate combination. For example, in leiomyosarcoma, the most prominent genetic alteration is chromosome 10 deletions that affect PTEN, but this may be a secondary alteration. Nonetheless, this was modelled by genetically inactivating Pten in the smooth muscle cells of mice, which led to leiomyosarcomagenesis114. Another recent mouse model introduced oncogenic Kras and mutant Trp53 into the muscle of mice; these changes were sufficient to generate high-grade sarcomas with myofibroblastic differentiation115, but KRAS is rarely mutated in human sarcomas. Sarcomas with simple karyotypes that have been successfully modelled are synovial sarcoma62,63, ARMS116, myxoid liposarcoma117 and GIST118,119. Nevertheless, in one model of synovial sarcoma62, the SYTSSX fusion oncogene was targeted to the myogenic lineage, a lineage that is inconsistent with conventional pathological data on thissarcoma. These and other sarcoma models may reveal the specific roles of genes that are altered in primary tumours and might allow the identification of secondary genetic or phenotypic events that are required for sarcoma progression and/or metastasis (FIG.3). Engineered animal models may also be adapted to invivo RNAi screens, as has been demonstrated in models of hepatocellular carcinoma and lymphoma120122 (FIG.3). This approach to exploring gene function would be especially powerful in soft-tissue sarcomas with complex genotypes and numerous chromosome aberrations. Genetically engineered animal models can also be used for diagnostic or prognostic biomarker discovery, drug testing and
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a
Sequencing (such as, point mutations and indels) Tumour samples
Rearrangements
Expression
Statistically principled assessment of drivers and passengers
Copy number alterations (such as, amplication and deletion)
genetics is a powerful approach to target discovery that is also being applied to other cancers by the US National Cancer Institutes Cancer Target Discovery and Development Network124.
Plasmid-based screening Loss-of-function RNA interference and/or gain-of-function ORFeome (pooled or arrayed formats)
F OR
shRNA expression plasmids
cDNA expression plasmids
Model systems In vitro Cell lines and short-term cultures In vivo Orthotopic xenogra s and GEMMs Ex vivo Tissue slices
f Phenotype readout
Fluorescence Barcode array Sequencing
g
Target identication Single-target validation in orthogonal models
Figure 3 | Models and functional genetics. High-throughput integrative genomics, increasingly dominated by second-generation sequencing, identifies abnormalities of Nature Reviews | Cancer sequence, structure, expression or copy number (a) in sarcoma genomes (notwithstanding epigenetic modification). In parallel, model systems (cultured cells, animal models or tissue slices) need to be generated from human tumours (b) and similarly be genomically profiled to confirm that they represent primary tumours in the retention of driver alterations (c). From tumour profiles, computational methodologies (dashed arrow) analyse the patterns of recurrence to distinguish likely driver from passenger alterations (d) (BOX2). These models can then be subjected to high-throughput functional genetic analyses, including both gain-of-function approaches (such as open reading frame (ORF) overexpression with pLX-Blast-V5 or similar cDNA expression vectors) and loss-of-function approaches (such as RNA interference using pLKO1-puro or similar short hairpin RNA (shRNA) plasmids) (e). These approaches can be applied either to all genes (to identify genotype-selective targets from those that are not) or to those identified by the integrative genomic and statistical methods. The readouts of these high-throughput methods, be it fluorescence, barcode arrays, or sequence read counts, provide data on one of a large number of possible phenotypes and can be analysed (f) to identify genotype-dependent vulnerabilities in sarcoma cells, identifying targets that can be validated in orthogonal models (g), a subset of which may be suitable for therapeutic intervention. This model of genomics-driven functional genetics focuses on rapid functional annotation of cancer genomes. GEMM, genetically engineered mouse model; indel, insertion/deletion.
drug resistance studies123. Indeed, the combination of sarcoma tumour profiles, sarcoma model systems that faithfully represent the alterations that are characteristic of their tumour type, and invitro and invivo functional
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Therapeutic avenues Despite the many advances in identifying genetic abnormalities in sarcoma and in elucidating their function, cytotoxic chemotherapy remains the standard of care for most locally advanced and metastatic sarcomas. However, complete surgical resection is the best hope for cure, and few patients with unresectable disease are curable by cytotoxic chemotherapy. Indeed, there are currently few specific genetic lesions in sarcoma that are direct targets of therapy, unlike epithelial cancers that harbour mutations that confer sensitivity to targeted inhibitors125. The exception among sarcomas is GIST, in which the KIT kinase inhibitor imatinib achieves a partial response or stable disease in approximately 80% of patients with advanced or metastatic GIST, often within days, with some patients now on therapy for 10years126. These responses to imatinib depend, however, on the specific site of mutation; tumours with activation loop mutations are generally insensitive. The response to imatinib has also been disappointing in patients with wild-type KIT and PDGFRA genotypes, despite KIT pathway activation. These findings lend support to a genotype-driven paradigm of kinase inhibition. This paradigm may apply across tumours of diverse histologies that share addiction to a particular mutated kinase. For example, the recent success of RAF inhibitors in BRAF-V600E-mutant melanoma suggests that responses may be elicited in other tumour types with a dependence on oncogenic RAF127,128, a possible therapeutic option for the approximately 1% of adult patients with GIST with BRAF-V600E mutation66. GIST notwithstanding, other common sarcomas have shown very little sensitivity to existing tyrosine kinase inhibitors (TKIs), including leiomyosarcoma, high-grade undifferentiated pleomorphic sarcoma (formerly termed malignant fibrous histiocytoma) and well-differentiated or dedifferentiated liposarcoma129,130. Nevertheless, kinase-directed agents have produced responses in certain translocation-associated sarcomas130,131 (TABLE2). Among these are responses to imatinib in dermatofibrosarcoma protuberans (DFSP) with collagen I1 (COL1A1)platelet-derived growth factor- (PDGFB) fusions, as well as in giant-cell tumours of the tendon sheath with collagen VI3 (COL6A3)colony-stimulating factor 1 (CSF1) fusions132,133. MET inhibitor responses are seen in ASPS and clear-cell sarcomas with ASPLTFE3 and EWS-activating transcription factor 1 (ATF1) fusions, respectively 38,134,135. ALK inhibitor responses are seen in inflammatory myofibroblastic tumours with ALK fusions136; and IGF1R antibody responses are seen in Ewings sarcoma with EWSFLI1 or EWSERG fusions 137139. However, in none of these instances have clinical responses proved to be as durable as the responses observed in patients with GIST that are treated with imatinib or other TKIs. In addition, patients with Ewings sarcoma have an approximately 1015% response rate to anti-IGF1R therapy, but in these tumours, and in
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Table 2 | Targeted agents that produce responses in sarcoma
Agent
Imatinib
Tumour types
GIST* Dermatofibrosarcoma protuberans Tenosynovial giant cell tumour and pigmented villonodular synovitis
Known alteration or recognized or proposed target

KIT and PDGFR mutations COL1A1PDGF fusions COL6A3CSF1 fusions KIT mutations Unknown ASPLTFE3 fusion VEGFR2 mutations, VEGF or its receptors Unknown RANK ligand ALK fusions ASPLTFE3 fusion EWSATF1 fusions IGF1R and VEGFR, and IGF2 overexpression EWSFLI1 or EWSERG VEGFR2 mutations, VEGF or its receptors ASPLTFE3 fusion Unknown
Approximate response rate

~80% High High ~8% Unknown Unknown ~15% High Moderate Unknown Unknown Unknown Unknown 1015% ~15% Unknown ~15% High
Refs
126 132,133 200 201 202 145 129 203 204 205 38,206 134,135,206 207,208 137139 73,129, 143,209 144 130 146148
Sunitinib
Imatinib-resistant GIST Desmoid tumour and deep fibromatosis Alveolar soft-part sarcoma
Sorafenib Denosumab Crizotinib MET inhibitor IGF1R antibody
Angiosarcoma Desmoid tumour and deep fibromatosis Giant-cell tumour of bone Inflammatory myofibroblastic tumour Alveolar soft-part sarcoma Clear-cell sarcoma Solitary fibrous tumour-haemangiopericytoma Ewings sarcoma Angiosarcoma Alveolar soft-part sarcoma Synovial sarcoma
Bevacizumab Cediranib Pazopanib mTOR inhibitors
PEComas and related conditions lymphangio- mTOR leiomyomatosis and angiomyolipoma
ALK, anaplastic lymphoma receptor tyrosine kinase; ASPL, alveolar soft part sarcoma locus; ATF1, activating transcription factor 1; COL1A1, collagen typeI1; COL6A3, collagen type VI3; CSF1, colony-stimulating factor 1; EWS, Ewing sarcoma breakpoint region 1; FLI1, Friend leukaemia virus integration 1; GIST, gastrointestinal stromal tumour; IGF, insulin-like growth factor; IGF1R, insulin-like growth factor 1 receptor; PDGFR, platelet-derived growth factor receptor; PEComa, perivascular epithelioid cell tumour; TFE3, transcription factor binding to IGHM enhancer 3; TSC2, tuberin; VEGFR, vascular endothelial growth factor receptor. *Dasatinib and sorafenib are also indicated for KIT-mutant GIST.
angiosarcomas, preclinical evidence would have predicted a greater response rate131,140,141. The reason for this discrepancy is unknown, although perhaps only a small proportion of patients have overtly IGF1R-dependent tumours that are possibly driven by high serum IGF1 levels, as has been proposed in non-small-cell lung cancer 142. Among some less common sarcoma subtypes, several targeted agents seem to be active (TABLE2). In addition, the identification of moderately or highly prevalent genetic abnormalities in some subtypes has suggested new possibilities for therapy. VEGFR-directed therapies such as bevacizumab and sorafenib are associated with approximately 15% response rates in primary and ionizing radiation-induced angiosarcoma 129,143, with these responses perhaps associated most closely with KDR mutation 73 . ASPS tumours are sensitive to VEGF-directed therapy such as cediranib or sunitinib144,145. On the basis of the reduced expression of tuberin (TSC2), perivascular epithelioid cell tumours (PEComas) and related conditions such as lymphangioleiomyomatosis and angiomyolipoma respond to mTOR inhibition 146148. As NF1 inactivation leads to aberrant MAPK and mTOR pathway activity149,150, the NF1 mutations and genomic deletions that have recently been observed in pleomorphic liposarcomas and myxofibrosarcomas74 may identify a broader range of patients
who might respond to either RAFMEK inhibitors or rapamycin and its analogues (rapalogues). In fact, using rapalogues in several complex subtypes could be justified on the basis of highly prevalent PTEN deletions, as in leiomyosarcoma114. However, preliminary results of a PhaseIII study of the mTOR inhibitor ridaforolimus indicated that progression-free survival was extended by only 3.1weeks after the completion of cytotoxic chemotherapy compared with the control group, suggesting limited utility of mTOR inhibitors in patients with sarcoma who are not first selected on the basis of their genomic abnormalities. The finding of frequent PIK3CA mutations in myxoid/round-cell liposarcoma74 (TABLE1) suggests that at least this molecular subset of patients might benefit from PI3K inhibitors; this is currently being tested in clinicaltrials. Finally, and although it is not strictly a targeted chemotherapeutic agent, trabectedin, a DNA minor groove-binding drug that is now approved in Europe for use in sarcomas, shows a substantial response rate in myxoid/round cell liposarcoma with FUSCHOP and EWSR1CHOP fusions and perhaps in additional translocation-associated sarcomas151,152. Although the precise function of trabectedin in sarcomas is unclear, it seems to involve alterations in transcription downstream of histone and transcription factor binding 153.
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Trabectedin sensitizes cancer cell lines to FAS-mediated cell death154, and sarcomas with intact nucleotide excision repair (NER) seem to be more sensitive to the drug than those with dysfunctionalNER155. Acquired and adaptive resistance. Malignancies, both epithelial and mesenchymal in origin, have remarkable similarity in their mechanisms of acquired and adaptive drug resistance. Resistance to TKIs is frequently acquired through second-site mutations, as in KITmutant GIST156158. For patients with metastatic disease, the median time to progression on first-line imatinib therapy is approximately 2years. Here, the nature of secondary KIT mutations depends on the location of the primary KIT mutation. For example, GISTs harbouring the more common and imatinib-sensitive KIT exon11 mutation tend to become resistant by acquiring a second-site KIT mutation in exon 11 rather than in exon 9. Resistance to broad-based second-line KIT inhibitors (sunitinib), arising on average within ~6months, can also develop through selection for double KIT-mutant resistant clones159. Another mechanism of resistance may involve alternative oncogenic pathways or rewiring of signalling networks, as experimental evidence suggests is the case for IGF1R inhibitors in rhabdomyosarcomas and Ewings sarcoma cell lines160,161. This adaptive resistance is consistent with the lack of IGF1R mutations observed in cancer types in which these therapies areactive. To circumvent these mechanisms of drug resistance, additional novel agents and strategies will be required. A strategy currently being used for imatinib-resistant chronic myelogenous leukaemia is the development of second-generation or third-generation inhibitors with kinase-binding affinity or binding to sites other than the kinase domain itself 162. The newer generation KIT inhibitors that are in preclinical development bind to the switch pocket domain of the protein, overcoming the resistance that is mediated by most combinations of KIT mutations observed in clinical samples163. However, the complexity of polyclonal resistance in patients with imatinib-resistant GIST suggests that a single nextgeneration drug is unlikely to inhibit all mutant clones in a given patient, and broader therapeutic strategies need to be considered. Strategies being examined in clinical trials include drug combinations that block specific heterodimerization of receptor tyrosine kinases or multiple levels in a single signalling pathway. One such example is an inhibitor of cell division cycle 37 (CDC37), a protein that links KIT to the chaperone heat shock protein 90 (HSP90); this inhibitor may potentiate KIT degradation without the potential toxicity that is inherent to inhibiting too many HSP90 client proteins164166. Other approaches warrant study, including polypharmacology (the simultaneous inhibition of multiple targets)167. Transcriptional target genes as a therapeutic target. Considering the therapeutic strategies aimed at the aberrant transcriptional proteins that drive translocation sarcomas, we note that transcription factors are
considered poorly druggable because their protein protein and proteinDNA interactions have historically been difficult to inhibit with small molecules. This view may be changing 168,169, and, in sarcoma, a notable example exists with a small molecule that disrupts a crucial interaction of the EWSFLI1 protein with RNA helicase A in Ewings sarcoma170. Nevertheless, the most promising current approach to discovering therapeutic targets in translocation sarcomas is identifying the targets of the chimeric transcription factor and focusing on those that encode known drug targets. The receptor tyrosine kinase MET has emerged as such a target in several sarcomas. MET is a direct transcriptional target of ASPLTFE3 in ASPS 38 , and apparently also of PAX3FOXO1 in ARMS36,171. In clear-cell sarcoma, EWSATF1 transactivates microphthalmia-associated transcription factor (MITF), which in turn directly activates MET transcription134,172. The fact that these fusion proteins upregulate MET has justified a PhaseII multi-institutional study of the MET inhibitor ARQ197 in patients with advanced clear cell sarcoma and ASPS. The transcriptional targets of the Ewings sarcoma fusion protein EWSFLI1 seem to affect multiple pathways, including NOTCH173, HedgehogGLI174,175, WNT-catenin176, transforming growth factor- (TGF)177,178 and possibly IGF1R179. The IGF1R pathway may be dysregulated by EWSFLI1 at several levels, including IGF1 upregulation and IGF binding protein 3 (IGFBP3) repression138,180,181. The IGF1R pathway is also transcriptionally upregulated by the PAX3FOXO1 fusion in ARMS34,35. These findings have in part provided the rationale for trials of IGF1R inhibitors in these sarcomas141,182,183. Finally, in myxoid/round cell liposarcoma, the FUSCHOP fusion oncoprotein forms a complex with nuclear factor-B inhibitor (NF-BI) at target promoters, thereby upregulating NF-B target genes 184. Thus, NF-B pathway inhibition, which reduces the viability of myxoid liposarcoma cell lines185, may represent a new therapeutic option in this translocationsarcoma. Alternative therapeutic approaches. The insensitivity of many sarcomas to existing systemic therapy is driving the exploration of agents that are aimed at new types of targets. Among these are HSP90 inhibitors, which have been studied in GISTs but not in other sarcomas. Other novel targets include BCL-2, phosphatases that are involved in feedback control of oncogenic pathways and key mediators of epigenetic regulation, including histone deacetylases, histone acetyltransferases and DNA methyltransferases. Epigenetic approaches may lead to the re-expression of pro-apoptotic molecules, which may induce apoptosis or senescence or which may render sarcomas sensitive to other agents, although this is speculation at present. Similarly, cell cycle regulators including CDK4 and CDK6 (REF.186) have proved to be attractive but refractory targets, whereas strategies to target components of the mitotic apparatus such as aurora kinases are actively under development. Targeting the p53MDM2 pathway with nutlins is promising in tumours with MDM2 amplification84
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Smoothened inhibitor GDC-0449 SHH ERBB family inhibitors Getinib Erlotinib Trastuzumab Panitumumab IGF1R inhibitor IMC-A12 PDGF-specic antibody IMC-3G3 Tyrosine kinase inhibitors Imatinib Sunitinib Dasatinib Cediranib Sorafenib KIT VEGFR2 VEGF inhibitors Bevacizumab PTC299
PTCH
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PDGF family PI3K inhibitors LY249002 BEZ235 XL765 XL147
-secretase complex PI3K class 1a -secretase inhibitors RO4929097 NICD RBPJ AKT inhibitors MK2206 Perifosine BAD BCL-XL MDM2 antagonist Nutlin-3 BCL-2
RAS RAF inhibitors PLX4032 RAF265 XL281 Sorafenib HSP90 inhibitors GLI
NF1
PTEN
RAF MEK inhibitors PD0325901 AZD06244 RDEA119 XL518 INK4 CCND CDK4 CDK inhibitors PD0332991 Flavopiridol mTOR inhibitors Sirolimus Temisirolimus Everolimus
RHEB
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AKT1
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HDAC HDAC inhibitors PXD101 PCI-24781 Panobinostat
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p53 YEATS4
BCL-2 inhibitors BH3 mimetics ABT-737 ABT-263 AT-101
Proliferation
G1/S progression
G2/M arrest
Apoptosis
Survival Predominantly amplied and/or overexpressed
Predominantly mutated
Predominantly deleted and/or underexpressed
Figure 4 | Pathways for targeted therapy in sarcoma. Diverse subtype-specific alterations imply that various signalling Nature Reviews | Cancer pathways function aberrantly in sarcomas. Abbreviated pathways include RASRAF, PI3K, mTOR, p53, cell cycle and survival, Notch and Hedgehog signalling, all of which are targeted by a growing list of specific therapies. A subset of nodes are coloured by their dominant alteration type (see key). Targeted agents (yellow boxes) include those in clinical use and those in preclinical or early phase development in sarcoma. BAD, BCL-2-associated agonist of cell death; CCND, cyclin D; CDK, cyclin-dependent kinase; EGFR, epidermal growth factor receptor; HDAC, histone deacetylase; HSP90, heat shock protein 90; IGF1R, insulin-like growth factor 1 receptor; NICD, NOTCH intracellular domain; NF1, neurofibromin 1; PDGFR, platelet-derived growth factor receptor; PTCH, Patched; RB1, retinoblastoma 1; RBPJ, recombination signal binding protein for immunoglobulin kJ region (also known as CSL); RHEB, Ras homologue enriched in brain; SMO, Smoothened; SSH, slingshot; TSC2, tuberin; VEGFR2, vascular endothelial growth factor receptor 2.
(predominantly well-differentiated and dedifferentiated liposarcomas). With an increasing array of agents to test against this rare group of cancers (FIG.4), internationalscale cooperative studies are paramount, as is ensuring that patients with sarcomas are included, along with more common cancers, in clinical trials of biologically relevantagents.
Future directions The diagnosis and treatment of patients with sarcoma is entering a period of rapid evolution. The dramatic drop in the cost of personal genome sequencing may alter the clinical and therapeutic course for patients
with sarcoma, as it is becoming technically possible to guide patient care by the analysis of the patients cancer and normal genome sequences, and this may also soon become practically feasible187. Over the next few years, the catalogue of mutations that drive all but the least common diseases will become known, thanks to large-scale efforts, such as TCGA and the International Cancer Genome Consortium, as well as others. To prevent sarcomas from lagging behind epithelial cancers in target discovery, it will be crucial to develop robust models of disease to allow the rapid functional annotation of the genetic abnormalities that are identified from both research and clinical sequencing.
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Competing interests statement
The authors declare competing financial interests. See web version for details.
DATABASES
National Cancer Institute Drug Dictionary: http://www.cancer.gov/drugdictionary bevacizumab | cediranib | imatinib | rapamycin | ridaforolimus | sorafenib | sunitinib | trabectedin
FURTHER INFORMATION
Marc Ladanyis homepage: http://www.mskcc.org/mskcc/html/54069.cfm Cancer Target Discovery and Development (CTD) Network: http://ocg.cancer.gov/programs/ctdd.asp Human ORFeome: http://horfdb.dfci.harvard.edu/ Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer: http://cgap.nci.nih.gov/Chromosomes/ Mitelman OMIM: http://www.ncbi.nlm.nih.gov/omim The Cancer Genome Atlas (TCGA): http://cancergenome.nih.gov The RNAi Consortium: http://www.broadinstitute.org/rnai/trc/ The Sarcoma Data Portal: http://cbio.mskcc.org/cancergenomics/sarcoma/
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Acknowledgements
The authors apologize to the many authors whose relevant work they were unable to cite here owing to space limitations. They thank N. Schultz for providing pathway expertise, C.D.M. Fletcher for critical reading, and M. Meyerson and C. Sander for advice and support. This work was supported in part by The Soft Tissue Sarcoma Program Project (P01 CA047179 to S.S., M.L. and C.R.A.) and the SPORE in Soft Tissue Sarcoma (P50 CA140146-01 to S.S., M.L., C.R.A. and B.S.T.).

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Advances in sarcoma genomics and new therapeutic targets

2011 Macmillan Publishers Limited. All rights reserved

LI1 G F1 TB PH 12 AC ZF1 SUZ JA F1 JAZ

Intermediate-complexity dedierentiated liposarcoma

Paired-end (or mate-paired) sequencing

Frequency Discovery platform

Structural ~25% rearrangements Amplification ~25%

100, 101 197

Epithelioid haemangioendothelioma Mesenchymal chondrosarcoma

WWTR1 CAMTA1 HEY1 NCOA2

Myxofibrosarcoma NF1 and pleomorphic liposarcoma Chondrosarcoma or chondroma IDH1 or IDH2

Deletions, point 811% mutations and indels Point mutations ~56%

548 | AUGUST 2011 | VOLUME 11

2011 Macmillan Publishers Limited. All rights reserved

Statistically principled assessment of drivers and passengers

Copy number alterations (such as, amplication and deletion)

shRNA expression plasmids

cDNA expression plasmids

2011 Macmillan Publishers Limited. All rights reserved

Known alteration or recognized or proposed target

Approximate response rate

Sorafenib Denosumab Crizotinib MET inhibitor IGF1R antibody

Bevacizumab Cediranib Pazopanib mTOR inhibitors

PEComas and related conditions lymphangio- mTOR leiomyomatosis and angiomyolipoma

2011 Macmillan Publishers Limited. All rights reserved

PDGF family PI3K inhibitors LY249002 BEZ235 XL765 XL147

HDAC HDAC inhibitors PXD101 PCI-24781 Panobinostat

BCL-2 inhibitors BH3 mimetics ABT-737 ABT-263 AT-101

Survival Predominantly amplied and/or overexpressed

Predominantly deleted and/or underexpressed

2011 Macmillan Publishers Limited. All rights reserved

13. 14. 15.

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Competing interests statement

VOLUME 11 | AUGUST 2011 | 557

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