Molecular Alterations in Pediatric Sarcomas: Potential Targets for Immunotherapy

Sarcoma (1998) 2, 77± 87

REVIEW

Molecular alterations in pediatric sarcomas: potential targets for immunotherapy

THERESA J. GOLETZ,1 CRYSTAL L. MACKALL,2 JAY A. BERZOFSKY 1 & LEE J. HELMAN 2

1M olecular Immunogenetics and Vaccine Research Section, M etabolism Branch & 2M olecular Oncology Section, Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, M D 20892, USA

Abstract Purpose/results/discussion. Recurrent chromosomal translocations are common features of many human malignancies. While such translocations often serve as diagnostic markers, molecular analysis of these breakpoint regions and the characterization of the affected genes is leading to a greater understanding of the causal role such translocations play in malignant transformation. A common theme that is emerging from the study of tumor-associated translocations is the generation of chimeric genes that, when expressed, frequently retain many of the functional properties of the wild-type genes from which they originated. Sarcomas, in particular, harbor chimeric genes that are often derived from transcription factors, suggesting that the resulting chimeric transcription factors contribute to tumorigenesis. The tumor-speci® c expression of the fusion proteins make them likely candidates for tumor-associated antigens (TAA) and are thus of interest in the development of new therapies. The focus of this review will be on the translocation events associated with Ewing’ s sarcomas/PNETs (ES), alveolar rhabdomyosarcoma (ARMS), malignant melanoma of soft parts (MMSP) (clear cell sarcoma), desmoplastic small round cell tumor (DSRCT), synovial sarcoma (SS), and liposarcoma (LS), and the potential for targeting the resulting chimeric proteins in novel immunotherapies.

Introduction An alternative consequence of chromosomal translocations is the generation of functional Chromosomal abnormalities are common in human chimeric genes. This scenario is most common in tumors with many malignancies exhibiting clonal solid tumors and usually involves unrelated genes. chromosomal aberrations.1 The identi® cation of tu- Often, these translocation events affect genes encod- mor-speci® c chromosomal translocations aids in di- ing transcription factors, thereby generating agnosis and serves as a prognostic indicator.2± 6 With chimeric transcription factors with properties of an increasing understanding of the effect these both genes (Table 1). The fusion proteins often events have on normal cellular processes, novel ther- exhibit the DNA-binding speci® city of one gene apies can be developed which have greater with the activation domain of the other gene. Such speci® city and ef® cacy. fusion proteins activate/repress transcription, exhibit Two major consequences of chromosomal rear- altered DNA binding speci® city or participate in rangements in tumors have been identi® ed: the acti- novel protein± protein interactions. Thus, they are vation of an oncogene, or the creation of a novel thought to play a critical role in the neoplastic oncogenic protein. First, translocations can result in transform ation process. the activation of genes located at or near the break- The identi® cation of translocations associated point. Often, these genes normally function in the with a group of primitive sarcomas, and the sub- promotion of cell growth and differentiation. Thus, sequent cloning of the chromosomal breakpoint re- their disruption can affect normal cell regulation. gions, has revealed that a common theme in these This type of alteration, which is most common in tumors is the generation of chimeric transcription hematological malignancies, is illustrated by the factors. The fusion proteins are expressed exclu- t(8;14) translocation associated with Burkitt’ s sively in the tumor cells, and function as potent lymphoma in which c-MYC is activated by reposi- transcription factors where they are thought to con- tioning under the control of the potent Ig enhancer.1 tribute to neoplastic transformation by mediating

Correspondence to: L. J. Helman, Molecular Oncology Section, Pediatric Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Building 10, Room 13N240, 9000 Rockville Pike, Bethesda, MD 20892± 1928, USA. Tel: 1 1 301 4964257; Fax: 1 1 301 4020575; E-mail: [email protected]. 1357-714 X/98/020077± 11 Ó 1998 Carfax Publishing Ltd 78 T. J. Goletz et al.

Table 1. Tumor-speci® c translocations associated with solid tum ors

59 /39 fusion Tumor Translocation product Type

Ewing’s sarcoma/ t(11;22)(q24;q12) EWS/FLI-1 RNA binding PNET t(21;22)(q22;q12) EWS/ERG ETS TF t(7;22)(p22;q12) EWS/ETV1 Alveolar t(2;13)(q35;q14) PAX3/FKHR PB and HD/FD rhabdomyosarcoma t(1;13)(p36;q14) PAX7/FKHR Melanoma of soft parts t(12;22)(q13;q12) EWS/ATF1 RNA binding/ (clear cell sarcoma) bZIP TF DSRCT t(11;22)(p13;q12) EWS/WT1 RNA binding/ Zn ® nger TF Synovial sarcoma t(X;18)(p11.2;q11.2) SYT/SSX1 SH2/KRAB box SYT/SSX2 Liposarcoma t(12:16)(q13;p11) CHOP/FUS-TLS RNA binding/ (myxoid and round cell) bZip TF

aberrant expression of normal genes. Several of the EWS encodes a 656-aa protein, the function of chimeric genes have been cloned and found to con- which remains unclear. W hile this protein is ubiqui- fer a transformed phenotype when expressed in tously expressed, expression levels ¯ uctuate with the vitro.7± 11 The tumor-speci® c expression of the fusion cell cycle.19± 23 EWS contains two major functional proteins make them likely candidates for tumor- domains. The ® rst is the N-terminal region (exons associated antigens (TAA), in which the junction 1± 7) consisting of a series of degenerate repeats that point creates a neo-antigenic determinant. The fo- resemble the transactivation domains of several cus of this review will be on the translocation events transcription factors, such as SP-124 while the se- associated with Ewing’ s sarcomas/primitive neu- cond region, the C-terminal region, includes a puta- roectodermal tumors (PNETs) (ES), alveolar rhab- tive RNA-binding domain (exons 11± 13) de® ned by domyosarcoma (ARMS), malignant melanoma of a conserved 80-aa domain.24 Wild-type EW S has soft parts (MMSP or clear cell sarcoma), desmo- been shown to bind RNA in vitro and EW S/GAL4 plastic small round cell tumor (DSRCT), synovial fusion proteins can activate a reporter gene, suggest- sarcoma (SS), and liposarcoma (LS), and the poten- ing a role for EW S in transcription.9,21,23 tial for targeting the resulting chimeric proteins in FLI1, a member of the ETS family of transcrip- novel immunotherapies. tion factors, is the human homologue of the murine FLI1 gene and is normally expressed in hematopoietic tissues.25 The ETS DNA-binding domain, usu- Tumor-associated chromosomal transloca- ally located in the C-terminal portion of the protein, tions in pediatric sarcomas is an 85-aa region that recognizes target genes through a conserved GGAA/T sequence.26 In FLI1, Ewing’ s sarcoma/primitive neuroectodermal tumors the ETS domain is encoded in the C-terminus, and The ES/PNET family of tumors is a group of poorly the N-terminal region contains a domain that is differentiated malignancies that include Ewing’ s sar- functional in reporter gene assays.9,27 coma (ES), peripheral neuroepithelioma (PNET) EWS/FLI1 is a potent transcription factor that and Askin’ s tumor. They are thought to originate can transform NIH 3T3 cells, and studies have from the neuroectoderm, and show varying, but shown that sequences in both EW S and FLI1 are limited degrees of neural differentiation. These tu- essential for transformation.7± 9 To better de® ne the mors express MIC2, a membrane protein that ap- functional regions of the fusion protein, substitu- pears to function in cellular adhesion. The tions were made in which domain 1 of EW S was expression of this antigen distinguishes these tumors replaced with a strong heterologous activation do- from other small round cell malignancies.12,13 In main. Many of these fusion proteins retained ac- addition, approximately 85% of ES/PNET tumors tivity, although not all were transforming.7,23 are characterized by t(11;22)(q24;q12).14± 17 Delat- Domain 2 of EW S could also be exchanged with a tre et al. demonstrated that the t(11;22)(q24;q12) weak transcriptional activation domain from TLS/ rearranges the FLI1 gene (Friend leukemia inte- FUS without loss of activity. Thus, these data sup- gration site 1) on chromosome 11q24 with a hereto- port a model wherein the EWS region of EWS/FLI1 fore uncharacterized gene, EWS.8,18 There is no confers strong transactivation through domain 1 evidence for the expression of the reciprocal hybrid with additional properties (protein± protein interac- transcript.19 tion) contributed by domain 2. Molecular alterations in pediatric sarcomas 79

Several variants of the t(11;22)(q24;q12) EWS/ cells expressing EWS/FLI1 and found that ex- FLI1 gene fusion have been described,8,19 but most pression of the fusion protein antisense RNA in- include EWS exons 1± 7 and FLI 1 exons 8 and 9.2,28 creased susceptibility to apoptosis. Thus, Therefore, the amino terminal portion of EW S is EW S/FLI1 may contribute to malignant transform- always fused to the carboxy terminal region of ation by alteration of more than one gene or gene FLI18,19 which suggests that these EWS/FLI1 vari- pathways. ants contribute to oncogenesis by similar mecha- The EWS gene is also involved in several other nisms. tumor-associated translocations. For example, a EWS/FLI1 and FLI1 have similar DNA-binding minority of PNETs present with a variant t(21; 22) speci® city and af® nities,9,29 but EWS/FLI1 is a more translocation that fuses EWS to the ERG gene.3,28,36 potent transactivator than FLI1.9,29,30 In vitro studies Like FLI1, ERG is a member of the ETS family of suggested that EW S/FLI1 functioned as a transacti- transcription factors and may regulate similar target vator at 10-fold lower concentrations than FLI1.29 genes.32 Studies are underway to identify ERG Thus, it is likely that EWS/FLI1 mediates its trans- target genes. Several lines of evidence suggest EWS/ forming effects, at least in part, by transactivation of ERG may contribute to neoplastic transformation FLI1 targets or promoters containing ETS-binding by the same or similar mechanisms as EWS/FLI1. sites. Because c-M YC is upregulated in some tu- First, PNETs containing EW S/FLI1 or EW S/ERG mors, including ES, one potential target gene of are phenotypically and clinically indistinguish- EW S/FLI1 was thought to be c-M YC. A study by able.2,36 As is seen in EWS/FLI1, EW S/ERG fusions Bailly et al. investigated transactivation of c-MYC by include EWS exons 1± 7, with ERG sequences en- EW S/FLI1 using transient tranfection HeLa cells. coding the ETS domain.3,28,36 The fusion protein These experiments suggested that EW S/FLI1 also functions as a transcription factor and requires played a role in increased expression of c-MYC. the same regions for transactivation de® ned in EWS/ However, direct binding of EW S/FLI1 to ETS- FLI1 studies.21 Furthermore, cells expressing EWS/ binding sites in the c-M YC promoter could not be ERG have a decreased ability to undergo apoptosis. detected using gel shift mobility assays. Thus, EWS/ These cells could be made susceptible to apoptosis FLI1 upregulates c-M YC, albeit by an indirect by the expression of EW S/ERG antisense RNA.35 mechanism yet to be elucidated.29 Therefore, it is likely that EWS/ERG fusions con- Recent studies suggest that EWS/FLI and FLI1 tribute to oncogenesis in a manner similar to EWS/ exhibit some differences in DNA-binding and pro- FLI1. tein± protein interactions.31 Therefore, it is possible A rare, third variant, t(7:22)(p22;q12) has been that EWS/FLI1 also contributes to transformation described37 in which EWS is fused to ETVI, the by activating genes not normally regulated by FLI1. human homolog of the murine ETS gene ER81. It is Studies are ongoing to identify the normal targets of likely that EWS/ETV1 contributes to malignant EW S/FLI1 and FLI1. Braun et al.32 utilized repre- transform ation by mediating aberrant transcription sentational difference analysis (RDA) to identify and/or repressing expression of regulatory genes. differentially expressed genes from NIH 3T3 cells However, RDA analysis of EWS/ETV1 revealed containing EW S/FLI1 or normal FLI1. This ap- that only one of eight EWS/FLI1 target genes was proach revealed that several transcripts were depen- upregulated by EW S/ETV1. This suggests that dent on the fusion protein for expression, while at EW S/ETV1 activates only a portion of the EWS/ least two transcripts were repressed. Stromelysin 1, FLI1 transform ation pathway, requiring other alter- cytokeratin 15, and a murine homolog of cy- ations for tumorigenesis, or that EWS/ETV1 plays a tochrome P-450 F1 are all induced following ex- minor role in transform ation. Further studies are pression of EWS/FLI1. However, the kinetics of needed to de® ne the effect of EWS/ETV1 on nor- expression argue against the direct upregulation of mal gene expression. all of these target genes. The elucidation of such Recently, Peter et al. identi® ed a new member of primary targets will provide insight into the role of the ETS family fused to EWS in Ewing’ s sarcoma, EW S/FLI1 in transformation. It is likely that the the FEV gene.38 FEV, which maps to chromosome oncogenic properties of EWS/FLI1 results from 2, encodes a 238-aa protein. Its expression is highly both the inappropriate expression of FLI1 target restricted with protein being detected only in adult genes, as well as novel protein± protein interactions prostate and small intestines, but not in other fetal which may lead to the activation of non-FLI1 target or adult tissues. FEV contains an ETS DNA bind- genes. Studies that utilized antisense EW S/FLI1 ing domain closely related to that of ERG and cDNA to diminish EWS/FLI1 RNA levels demon- FLI1; however, in contrast to these proteins, FEV strated markedly decreased cell growth in vitro, has a small N-terminal region of only 42 aa which thereby implicating the fusion protein as a key con- suggests that it lacks important transcription regula- tributor to aberrant growth.33,34 EWS/FLI1 may tory domains present in other ETS family proteins. contribute to oncogenesis is by inhibition or alter- It is unclear whether or not EWS/FEV alters tran- ation of normal apoptotic pathways. Yi et al.35 ob- scription of similar target genes than other EW S served suppression of apoptosis in Ewing’ s sarcoma fusion proteins. Further studies are needed to 80 T. J. Goletz et al. elucidate this fusion protein’ s role in the pathogene- less than wild-type PAX3, the fusion protein is a sis of ES. more potent transactivator.46± 49 Overexpression of The common denominator of these tumors is that murine PAX3 transforms NIH 3T3 cells11 and the all are primitive neuroectodermal sarcomas occur- PAX3/FKHR fusion protein transformed chicken ring in children and young adults, and the evidence embryo ® broblasts.10 One possible mechanism of strongly implicates EWS fusions as key mediators of transform ation is through a gain of function, not malignant transformation. There is also strong evi- only by increased transactivation potency, but also dence to suggest that these fusion proteins contrib- through constitutive and increased expression.49,50 ute to oncogenesis by aberrant expression of target Interestingly, a recent study which utilized antisense genes (activation and repression), as well as altering technology to downregulate PAX3/FKHR in ARMS the expression of genes not normally regulated by tumor cells demonstrated reduced cell viability, the native transcription factors.32 Furthermore, which led to the conclusion that PAX3/FKHR may these genes may effect normal growth regulation by contribute to malignant transformation through interfering with apoptotic pathways.35 suppression of apoptotic processes which would normally cause cell death.51 Interestingly, 10± 20% of ARMS tumors contain a Alveolar rhabdomyosarcoma (ARMS) variant translocation, t(1;13)(p36;q14), that results Rhabdomyosarcoma is the most common soft tissue in the in-fram e fusion of 59 PAX7 to 39 FKHR. sarcoma in pediatric patients, with approximately PAX7 and PAX3 are highly homologous in the PB 250 cases per year in the United States. Roughly and HB domains, suggesting that they might recog- 20% of these cases are of the alveolar morphological nize similar target genes.40± 43,52 Furthermore, the type (ARMS) which is characterized by alveolar-like PAX3/FKHR and PAX7/FKHR chimeric proteins spaces formed by ® brovascular septa. These spaces share structural similarities in that they both contain are ® lled with malignant cells that are distinguished intact N-terminal PB and HB regions fused to the by their eosinophilic cytoplasm . Approximately 80% acidic and proline-rich C-terminal region of of ARMS express a translocation involving the long FKHR.41,42,52 Therefore, it is likely that these arms of chromosomes 2 and 13 t(2;13)(q35;q14), translocations create similar chimeric transcription which results in the juxtapositioning of a truncated factors that contribute to transform ation by altering PAX3 gene of chromosome 2 to the 39 -terminal expression of a common group of target genes.50± 52 region of the FKHR gene of chromosome 13.39± 43 The PAX family of transcription factors play im- Malignant melanoma of soft parts (M MSP) or clear cell portant roles during embryonic development, partic- sarcoma (CCS) ularly in morphogenesis and pattern formation.44 These genes contain a paired-box (PB) DNA- Malignant melanoma of soft parts (MMSP), also binding domain and some also contain a homeobox known as clear cell sarcoma (CCS), is a rare, but (HB) DNA-binding domain. Overexpression of aggressive soft tissue sarcoma of muscle tendons and these genes can result in oncogenic transform- aponeuroses that occurs most frequently in young ation10,11 and loss of function mutations has been adults between the ages of 15 and 35 years.53 Over observed in several genetic diseases, including 95% of MMSP cases occur in the extremities, and W aardenburg syndrome.45 only rarely (less than 2%) occur in the head and FKHR, formally known as ALV,41 is a member of neck region. Although MMSP is a melanin-produc- the fork-head domain (FD) family of transcription ing tumor, there is no evidence to suggest that these factors which contain a conserved DNA-binding tumors are directly related to malignant melanoma. motif related to the Drosophila region-speci® c home- MMSP is thought to have neuroectodermal origins54 otic gene fork-head. This family of transcription and expresses neural antigens, as well as markers of factors normally functions during embryogenesis. melanin production, such as HMB-45. A t(12;22) The FKHR gene is ubiquitously expressed and func- (q13;q12) translocation event is present in more tions as a transcription factor. than 70% of these tumors55,56 and molecular analy- The hybrid gene which results from the sis of the breakpoint reveals an EWS/ATF1 fusion. t(2;13)(q35;q14) translocation encodes a fusion This chimeric protein joins the 59 RNA-binding protein containing the amino terminal portion of the region of the EWS gene and the 39 region of the PAX3 protein including the PB and HB domains ATF1 gene, a member of the CREB/transcription joined to the carboxyl region of the FKHR protein factor family of leucine zipper transcription factors that is truncated within the winged helix DNA- that has a bZIP domain for DNA binding and binding region, but retains a putative transactivation protein± protein interaction.57 This family of tran- domain. Evidence suggests that the DNA-binding scription factors mediates transcription through speci® cty of PAX3/FKHR is contributed by PAX3, ATF-binding sites. The expression of these genes is most likely through the PB and HB domains, while induced by cAMP, and they are activated by phos- FKHR contributes the transactivation region. Al- phorylation by cAMP-dependent protein kinase A though the DNA-binding activity of PAX3/FKHR is (PKA).58,59 Molecular alterations in pediatric sarcomas 81

The t(12; 22) translocation fuses the N-terminal Synovial sarcoma (SS) portion of EWS to the C-terminal region of ATF1, Synovial sarcoma is an aggressive soft-tissue malig- retaining the bZIP domain. However, the PKA reg- nancy which occurs primarily in the extremities near ulatory phosphorylation site is lost.58 Thus, it is major joints (e.g. ankle, knee) of adolescents and likely that EW S/ATF1 could exhibit the DNA-bind- young adults. Virtually all synovial sarcomas contain ing speci® city of ATF1, and dimerize with CREB, 68 a translocation of chromosomes X and 18 with but would not be cAMP-inducible. EW S/ATF1 approximately 70% involving t(X;18)(p11.2;q11.2). does activate promoters with ATF1 binding sites, This translocation event generates a fusion protein although not all such promoters were activated,60 from the 59 region of the SYT gene and the 39 and some promoters were found to be repressed by 69± 71 region of SSX1 or SSX2. There is no evidence EW S/ATF1. Therefore, EW S/ATF1 may contribute of a transcript being expressed by the reciprical to malignant transform ation by several mechanisms. hybrid der (18).71 The function of the SYT gene is First, EW S/ATF1 may constitutively activate ATF1 unknown, and sequence analysis reveals no classical target genes that are normally induced by cAMP, or structural motifs associated with DNA-binding or it may repress genes that normally function in transcriptional regulation. However, the presence of growth control. Alternatively, EWS/ATF1 may acti- SH2 and SH3 domains suggests that SYT might vate novel genes, perhaps genes regulated by other function through protein± protein interaction. The CREB/ATF family members. recent isolation of the mouse homolog of SYT re- In most MMSP tumors, two hybrid transcripts vealed that SYT is expressed ubiquitously during are generated and expressed by the t(12;22) 69 early embryogenesis, but expression is restricted (p13;q12) translocation. The expression pro® le of later in development to cartilage tissue, speci® c neu- the fusion gene on der(12) chromosome is compat- ronal cells and some epithelial-derived tissues. SYT ible with the ubiquitous expression of ATF. How- was also detectable in primary spermatocytes. ever, this out-of-fram e fusion results in a product Several studies suggested that SS contained two consisting of the ® rst 65 N-terminal amino acids of distinct X chromosome breakpoint sites. However, ATF1, which is unlikely to bind DNA or dimerize, the identi® cation of two closely related genes at making its role in transform ation unclear. It is un- Xp11.2 established the involvement of distinct cod- likely that expression of the der(12) transcript is ing regions. Despite being 2 Mb apart, SSX1 and essential in transform ation given reports that 30% of 70 SSX2 share 80% homology. Both encode a 188-aa MMSP lack expression.56 protein with an N-terminal Kruppel-associated box (KRAB) that is thought to function as a transcrip- 72,73 Desmoplastic small round cell tumor (DSRCT) tion repressor domain. Although these proteins lack zinc ® nger motifs, the presence of the KRAB Desmoplastic small round cell tumor (DSRCT) is sequences suggest a role in transcription. However, an aggressive small round cell tumor that occurs this domain is not present in the chimeric protein, predominantly in abdominal serosal surfaces and which suggests that SSX1 and SSX2 sequences has a predilection for young males.61 The tumor is a contribute to transformation through novel protein± primitive small round cell with features of divergent protein interactions or some other function. SSX3, differentiation, co-expressing epithelial, neural and another KRAB protein, is not implicated in t(X; myogenic markers. The origin of this tumor remains 74 18)-positive SS, but has high homology to SSX1 unclear, but it is most likely derived from the and SSX2 (95 and 90%, respectively). The study of mesothelium. Almost 100% of these tumors contain this gene may provide insight into the function of a t(11;22)(p13;q12) translocation that fuses the 59 SSX1 and SSX2. region of the EWS gene to the 39 region of WT1, a tumor suppressor gene involved in a subset of 62± 66 Liposarcomas (LPS) W ilms’ tumors. W T1 binds DNA through a series of zinc ® ngers and represses the transcription Liposarcomas (LS) are soft tissue tumors that occur of certain genes. These zinc ® ngers are essential for primarily in the extremities and retroperitoneum. transcriptional repression. The chimeric protein These tumors are from primitive mesenchymal cells contains the N-terminal region of EWS fused to the and they resemble fetal adipose tissue. Several W T1 DNA-binding domain. Given that both the characteristic cytogenetic aberrations have been wild-type EWS gene and EWS fusion proteins are identi® ed for adipose tumors. The most common known to participate in transcriptional complexes, it LS are myxoid round cell liposarcomas, and greater is likely that EWS/WT1 functions as a transcription than 90% of myxoid liposarcomas contain the factor, possibly through WT1 targets. Therefore, t(12;16)(q13;p11) translocation in which CHOP unlike the loss of function mutation in W ilm’ s tu- on the long arm of chromosome 12 is fused to mor, the loss of the zinc ® nger region of WT1 in FUS/TLS.22,75± 77 However, this translocation event EW S/WT1 serves to convert WT1 from a repressor has not been detected in other adipose tumors and, of transcription to a dominant transcriptional activa- therefore, may provide interesting insight into the tor oncogene.67 transform ation process of this subset of tumors. 82 T. J. Goletz et al.

FUS/TLS is structurally similar to EWS ( . 50% and recognition has increased considerably in the amino acid identity)75 and is expressed at high levels last two decades and has been expertly reviewed in all tissues examined.22 TLS binds RNA and elsewhere.81 Brie¯ y, T cells recognize antigens as encodes a strong transcriptional activation domain short peptides that are bound to the cell surface in in the N-terminal region.78 Therefore, like EW S, the context of major histocompatibility (MHC) FUS/TLS may function as a nuclear RNA-binding molecules.81,82 In the case of CD8 1 CTL, the T cell protein. receptor (TCR) recognizes short peptides (8± 10 CHOP, also called GADD153, is a member of amino acids) bound to MHC class I molecules. the CCATT/enhancer-binding protein (C/EBP) These peptides are derived from endogenously ex- family of leucine zipper transcription factors that pressed proteins which undergo proteolytic process- regulate adipocyte differentiation. CHOP is ex- ing in the cytosol by large proteosome complexes. pressed at low levels in adipocytes; however, mRNA Peptide fragm ents are then transported into the levels increase during conditions of stress such as lumen of the endoplasmic reticulum (ER) by spe- DNA damage. Overexpression of CHOP in NIH cialized transporters of antigen processing (TAP). 3T3 cells results in growth arrest at G1/S.79 Thus, Once inside the ER, peptides associate with an CHOP is thought to function as a dominant nega- appropriate MHC class I molecule that is associated tive growth regulator.80 with beta-2-microglobulin (b 2 m), an invariant sub- In the TLS/CHOP fusion protein, the N-terminal unit which is thought to enhance ef® cient MHC portion of TLS is joined to the entire CHOP coding folding, optimize MHC/peptide binding, and in- region.75,76 TLS/CHOP can transform NIH 3T3 crease stability of the MHC/peptide complex during cells and studies indicate that transformation re- transport to and expression on the cell surface. quires sequences from both TLS and CHOP.78 The Following peptide/MHC binding, the peptide/ requirement for the C-terminal leucine zipper do- MHC/b 2 m complexes transverse the ER and Golgi main of CHOP for transform ation suggests a crucial apparatus, and are displayed on the cell’ s surface role for C/EBP protein dimerization. Although it is where they are subject to surveillance by CTL. In unclear whether normal wild-type CHOP activation the case of CD4 1 Th cells, the TCR recognize requires DNA-binding, the potential DNA-binding slightly larger peptides (10± 25 aa) in the context of region, a basic region of the bZIP domain, is re- MHC class II molecules. These peptides are typi- quired for transform ation. The role of TLS se- cally derived from material or organisms which have quences in transformation may be more than that of undergone endo/phagocytosis by APC. Thus, in a strong transactivator, since substitution of this general, CD8 1 CTL recognize intracellular (en- region with other potent transactivating domains did dogenous) peptides while CD4 1 T cells recognize not mediate transformation. However, substitutions external (exogenous) protein fragm ents. with EWS sequences were transforming.78 There- CTL can distinguish self from non-self peptides fore, TLS/CHOP may contribute to transformation associated with MHC class I molecules, so that by mechanisms similar to those previously discussed expression of viral proteins or altered cellular in EWS fusion proteins. proteins will be re¯ ected in the peptide/MHC complexes displayed on the cell surface. Although the tumor-speci® c fusion proteins described in this re- Potential immunotherapeutic approaches for view function as nuclear transcription factors, they the treatment of pediatric sarcomas are still subject to the proteolytic processing and Although multi-modality therapy has improved sur- presentation pathways described. There is exper- vival rates for the pediatric sarcomas described in imental evidence that tumor-associated nuclear this review, patients often relapse, at which time proteins, such as mutant p53, can induce immune responses to multi-agent chemotherapy are brief or responses.83± 88. non-existent. Furthermore, patients who present The identi® cation of TAA and an increased with metastatic disease at diagnosis do very poorly understanding of the requirements for the induction in spite of aggressive multi-modality therapy. There- of cell-mediated immune responses (Table 2) has fore, efforts are needed to develop novel treatments, led to advances in immunotherapy.89 While a num- such as immunotherapies. Studies over the past ber of TAA have been identi® ed for several tumor decade have provided evidence that treatments types,90± 93 it is unclear whether all TAA will be based on the manipulation of the immune system effective tumor regression antigens. Ideally, one can mediate regression of established metastatic would like to identify and target TAA which play a cancer. More speci® cally, cell-mediated immunity key role in neoplastic transformation, so that they can play a critical role in tumor regression. cannot be lost without loss of malignancy. The T lymphocytes are most often categorized as tumor-associated translocations identi® ed for a CD8 1 cytotoxic lymphocytes (CTL) or CD4 1 number of pediatric sarcomas such as ES and AR helper lymphocytes (Th), and both types of T cells may very well be such antigens, since they generate are known to play a role in tumor regression. Our functional chimeric transcription factors known to understanding of antigen processing, presentation, contribute to abberrant gene expression. More Molecular alterations in pediatric sarcomas 83

Table 2. Immunotherapeutic approaches using tum or-associated antigens

Active immunotherapy using immunodominant peptides: alone with adjuvants linked to helper peptides Administered: in lipids/liposomes pulsed onto antigen-presenting cells (APCs) Substituted peptides immunodominant peptides with amino acid substitutions to increase binding to MHC Proteins alone with adjuvants DNA `naked’ DNA encoding cancer antigens administered using gene gun intramuscular injection associated/linked to lipids Recombinant viruses recombinant viruses, such as vaccinia, fowlpox or adenovirus, encoding cancer antigens, alone or in combination with genes encoding cytokines costimulatory molecules or immunostimulatory factors Recombinant bacteria recombinant bacteria such as bacillus calmette± guerin (BCG), Salmonella or Listeria engineered to express cancer antigens alone or with genes encoding cytokines, costimulatory molecules or other immunostimulatory factors Active immunotherapy followed by cytokines Interleukin 2 (IL-2), IL-6, IL-10, IL-15 Passive immunotherapy with anti-tumor lymphocytes generated in vitro Generation of CTL using immunodominant peptide-pulsed APCs Generation of Th by coincubation of APC with antigenic peptides

speci® cally, the breakpoint junctions are likely neo- does not react against all possible antigenic determi- antigens. Further, it should be possible to avoid nants, characterization of the immunodominant autoimmune responses by focusing on minimal pep- peptides in the tumor regression antigens will fur- tides corresponding to the sequences which span the ther aid in the development of effective treatments.94 breakpoint, since these would not be present in The identi® cation of TAA and the cloning of the normal cells. This hypothesis was tested in animal genes which encode them provides numerous op- models using synthetic peptides corresponding to portunities for the development of cancer therapies the breakpoint junctions in ES and ARMS as im- (Table 2). Therapies could utilize the TAA protein munogens. In these studies, peptide-pulsed APC either alone or with adjuvants. Alternatively, the administered intravenously, generated CD8 1 CTL administration of peptides derived from the TAA responses capable of lysing peptide-pulsed tumor protein administered alone, with adjuvants or in cells in vitro as well as tumor cells transfected to combination with helper peptides, has certain ad- express the full-length fusion protein. Furthermore, vantages in that this approach has been demon- these responses were able to reduce or irradicate strated to generate T cell responses while having tumor in vivo. These data demonstrate that the minimal risk in the induction of unwanted and chimeric fusion products resulting from chromo- potentially dangerous autoimmune reactions. Anti- somal translocations can serve as neoantigens. tumor responses generated by peptide vaccination Because the translocation events are tumor speci® c, may be augmented by manipulation of the route/ therapies targeting the resulting fusion proteins mode of administration. The cloning of genes en- would be highly speci® c and potentially less toxic. coding TAA will facilitate their expression in Clinical trials are currently underway in patients high-ef® ciency expression systems, such as recombi- with ES and ARMS to evaluate the generation of nant viruses or bacteria. These vectors can be engi- anti-tumor responses using a similar approach. In neered to express the TAA alone or in conjunction addition, studies are ongoing to not only identify with cytokine genes or genes encoding costimula- additional TAA, but also to gain an understanding tory molecules. Furthermore, direct injection into as to which TAA may serve as tumor rejection muscle of DNA encoding antigens or the use of antigens. Since it is clear that the immune system `gene guns’ in which DNA is attached to small 84 T. J. Goletz et al. particles that are mechanically propelled into cells promoter region from one of the partner genes. is also an effective method of inducing immune However, this mechanism has not been observed in responses.95± 100 solid tumors, but may be relevant in hematopoietic Anti-tumor responses have been generated by in malignancies. Nonetheless, it is likely that ex- vitro sensitization of peripheral blood lymphocytes pression of hybrid proteins in solid tumors dysregu- (PBL) to peptide-pulsed APC or irradiated tumor lates the transcription of key growth control genes or cells. Repeated in vitro sensitization using im- pathways, thereby promoting tumorigenesis. munodominant peptides from melanoma antigens While fusion proteins are likely to invoke a combi- pulsed onto autologous peripheral blood mononu- nation of the aforementioned mechanisms, the re- clear cells in the presence of IL-2 resulted in the dundancy of their role in oncogenesis is noteworthy. expansion of CTL (10,000-fold) over a 6-week pe- The multiple interchange of functional domains riod. Cells generated by this approach showed im- from related genes such as FLI1 and ERG in mune reactivity 50± 100 times greater than PNETs, PAX3 and PAX7 in ARMS and SSX1 and corresponding tumor in® ltrating lymphocytes SSX2 in SS result in similar tumor phenotypes.78 (TIL)101 and speci® cally recognized the appropriate Domain-swap experiments involving EWS for TLS immunodominant peptide as well as tumor cells as in TLS/CHOP showed that substitutions can be measured by lysis and cytokine release. Studies in made with little change in morphology. However, experimental animal models suggest that speci® c other experiments in which FLI1 was exchanged for tumor recognition as determined by lysis and cy- CHOP in fusions with TLS or EWS had an effect tokine secretion assays correlated highly with in vivo on cell morphology, such that the morphology in anti-tumor effects.102 These correlates have also some cases was dependent on the DNA-binding been observed in patients treated with autologous region of the chimeric transcription factor. Finally, TIL.103,104 In several other studies, T cells stimu- of note is the early onset of many of these tumors. lated in vitro were capable of recognizing and lysing This suggests that the genes involved in sarcoma-as- target cells pulsed with peptides known to bind to a sociated translocations have speci® c patterns of de- particular MHC class I molecule; however, these velopmental regulation, and that dysregulation of same T cells were often incapable of recognizing this temporal regulation has profound effects. and lysing the low levels of processed peptides ex- Attempts at developing new therapeutic ap- pressed by tumor cells.105 Thus, there is consider- proaches to the treatment of these tumors have able heterogeneity in anti-tumor responses. included immunotherapy. However, successful immunotherapeutic stratagies must meet several cri- teria, the ® rst of which is the expression of TAA that Summary are recognized by T lymphocytes. In the case of the The generation of chimeric transcription factors is a sarcomas presented in this review, the chimeric common consequence of chromosomal transloca- transcription factors represent potential TAA. Stud- tions in solid tumors. The resulting fusion proteins ies in experimental animals suggest that the translo- have been shown, in several cases, to have trans- cation breakpoints in ES and ARM S represent forming activity. Chimeric oncoproteins may func- neoantigens which can be recognized by CTL. Fur- tion through several mechanisms. First, a strong thermore, these response were suf® cient to mediate activation domain from one gene may be fused to in vivo tumor regression in animal models. Clinical the DNA-binding speci® city region of another gene, vaccine studies are ongoing to evaluate the ability of leading to dysregulated expression of target genes. these TAA to serve as tumor regression antigens. The fusion proteins associated with MMSP, ARMS, Finally, identi® cation of the immunodominant epi- and PNETs are examples of this mechanism. How- topes in tumor regression antigens will favor the ever, in myxoid liposarcoma, the FUS/CHOP gene induction of effective anti-tumor responses. Screen- product appears to mediate its effect on transcrip- ing vaccines and various delivery systems (peptides tion through protein± protein interactions and may or proteins in adjuvants or on dendritic cells, DNA, not require DNA-binding. Second, a fusion partner viruses) in animals, such as HLA-transgenics, will may contribute more than an activation domain. help to identify the most promising vaccines for use For example, the EWS/FLI1 fusion protein of ES in clinical trials. seems to combine the transactivation domain of EW S with the DNA-binding region of FLI1. How- ever, the fusion protein appears to mediate novel References protein± protein/protein± nucleic acid interactions. 1 Rabbitts TH. 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