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Molecular Alterations in Pediatric Sarcomas: Potential Targets for Immunotherapy

Molecular Alterations in Pediatric Sarcomas: Potential Targets for Immunotherapy Pu rpose/r esults/di scussion. Re cur rent c hromo som a l t rans locatio ns a re com m o n f eatur es o f ma ny h uma n m a lignanc ies. While s uch t ransloc ations o ft en s erve a s d iagnos tic m a rkers, m o lecular analysi s o f these b reakpoi nt r egions a nd t he characte rization o f the a ffected gene s i s l eadi ng t o a g reater und erstand ing o f the c ausal r ole s uch t ransloc ations p lay i n ma lignant t ransfor ma tion. A c omm o n t heme that i s e me rging from t he s tud y o f tum o r-assoc iated t ransloc ation s i s t he generati on o f chime ric gene s t hat, w hen e xpresse d, frequent ly r etain m a ny o f the f unc tional p roperti es o f the w ild-typ e genes f rom w hich they o r iginated . S arcoma s, i n p articul ar, h arbor c hime ric gene s t hat a re often d erived fr om t rans cription factors, suggest ing t hat t he r esul ting chime ric transcri ption fa ctor s c ont ribut e t o t um o rigenes is. T he tumo r-speci ® c expressi on o f the fu s ion p roteins m a ke t hem l ikely c andi dates fo r tum o r-assoc iated a ntigens ( TAA ) a nd a re thus o f int erest in t he d e velopme nt o f ne w therapie s. T he focus o f this r eview will b e on t he t ransloc ation e vents a ssociat ed w ith E wing’ s sarcoma s/P NETs ( ES), a lveol ar r habdom y osarcom a ( AR MS ), m a ligna nt m e lanoma o f soft p arts ( MM S P) (clear c ell sarcoma ), d esmo plastic sma ll r oun d c ell t um o r ( DSRCT ), synovia l s arcoma ( SS) , and l iposarc oma ( LS ), a nd t he p otent ial for targeting the r esul ting c hime ric proteins i n n ov el i mm u n ot herapies . Int roduc tion An a lter na tive c o ns equen ce o f ch romosomal trans loca tions is the g e ne ration o f funct iona l Chromos omal abnormalities are com m o n i n h uma n ch imer ic g e n es . Th is sce na rio is mo st c om m o n i n tum o rs with m a ny ma lign anc ies e xh ibiting c l on al solid tum o rs and u s ually inv olves un rel ated g e n es . ch romos oma l aberrations . Th e i de nt i® c ation o f tu- Of ten, the se t ransloc ation e ven ts a ffect g e ne s en c od- mo r -speci® c c hr omo somal trans location s aids in d i - ing t rans cr iption f actor s, ther eb y ge ne rating 2± 6 agn o sis and s erves as a progn os tic i ndi cator. With ch imer ic t rans cr iption f actors with p roperties of an inc reasing u n d e rstand i ng o f the e f fect t hes e both g e ne s (Ta ble 1 ) . T h e f usion protei ns often ev ent s have on n or mal cel lul ar proce sses, no v el t her - ex hi bit t he D N A -binding s peci ® c ity o f on e g e ne apies c an b e d e velope d w hich have gr ea ter with t he a ctivation d o ma in of the o ther ge ne . S uc h speci® c i ty a nd e f ® ca cy. fusion protei ns act ivate/ r epres s trans cr iption, ex h ibit Tw o m a jor con sequen c es of ch romos omal rear- alter ed D N A bindi ng s peci® c i ty o r partici pate i n rangem e nt s in t um o rs have been i de n ti® e d : the a cti- no vel p rotein± protei n i nt eracti on s . Th us, the y a re vation o f an on c og en e , o r the c reatio n o f a no vel thou g ht t o p lay a cr itical r ole i n t he n e o plastic on c og en i c p rotei n. Fi rst, trans location s can r es ult i n trans formation p roce ss. the a ctivation o f ge ne s located a t o r nea r the b reak- Th e i de nt i® c a tion o f trans loca tions associated point . O f ten, the se g e n es nor mally func tion i n t he with a group of primitive s arcoma s, and t he s ub- promo tion o f cel l g r owth a nd d i fferentiation . Th us, sequen t c lon i ng o f the c h romos oma l breakpoint r e- the ir d isruption c an a ffect n o r mal ce ll r egu l ation . gi on s, has rev ealed t hat a co mm o n t heme i n t hes e Th is type o f alterat ion, which is mo s t com m o n i n tum o rs is the g en e ration o f chi me ric t rans cription hema tolog i cal m a lign a nci es , is illus trated b y the factors. Th e f usion p roteins are ex pr es sed e xc lu- t(8; 14 ) t rans loca tion a ssociated w ith B u rkitt’ s sively in t he t um o r ce lls, and f unc tion a s poten t lymphoma in w hich c-MYC is activated b y repos i- trans cription f actors where t hey a re thou g ht t o c on- tion i ng u n de r the c o nt rol o f the p otent I g e nh ance r. tribut e t o n e o plastic t rans formation b y me d i ating Co rrespon dence t o: L. J . Helman, Mo lecular O n cology S ection, Pediatri c B ranch, Di vision o f C l inical S ciences , National C a ncer I nstitute , Na tional I nstitute s of H ealth, B uildi ng 1 0, Room 13N240, 9000 Rockvill e P ike, B ethesda, M D 2 0892± 1928, USA. Tel: 1 1 301 4964257; Fax: 1 1 301 4020575; E-ma il: h elmanl@p bmac.nci.ni h.gov. 1357-714X/ 9 8/020077± 11 Ó 1998 Ca rfax P ublishing L t d 78 T. J. Goletz et al. Table 1. Tumor-speci® c translocations associated with solid t umors 59 /39 fusion Tumor Transloca tion product Type Ewing’ s s arcoma / 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 rhabdo my osarco ma t(1;13)(p36;q14) PAX7/FKHR Me lano ma o f soft parts t (12;22)(q13;q12) EWS/ATF1 RN A b indi ng/ (clear c ell s arcoma ) bZIP TF DSRCT t(11;22)(p13;q12) EWS/WT1 RN A b indi ng/ Zn ® nger TF Syno vial s arcom a t(X;18)(p11.2;q11.2) SYT /S SX1 SH2 /K R A B b ox SYT/SSX2 Liposar coma t(12:16)(q13;p11) CHOP/FU S -TLS R N A b indi ng/ (my xoid a nd r ound c ell) bZip TF aberrant e xp res sion of no rmal ge n es . Sev eral of the EWS en c od es a 65 6 -aa protei n, the f unct ion o f ch imer ic g e ne s have bee n c lon e d a nd f ound t o c on - which r em a ins un cl ea r. While t his protein i s ubiqui- fer a transformed p hen ot ype w hen e x pr es sed in tou sly exp ressed, exp ression l evel s ¯ u c tua te w ith t he 7± 11 19± 23 vitro. Th e t um o r -speci® c e xp ression o f the f usion cell cycle. EWS con tains two m a jor funct iona l proteins ma ke them likely ca ndi da tes f or tum o r - do ma ins. Th e ® rst is the N - ter mina l regi on ( ex o ns associated a ntige ns (TA A), in w hich the j unc tion 1± 7) c o ns isting o f a ser ies of de ge n er ate r epea ts tha t point c reates a ne o- antige n i c d e termi na nt. T h e f o- resemb le t he t rans activation d o m a ins of sever al cu s of this review will b e o n t he t rans loca tion e ven ts trans cription f actors, such a s SP- 1 while t he s e- associated w ith E w ing ’ s sarcoma s/primitive n e u - co nd r egi on, the C -termi na l regi on , inc lude s a put a- roec tod er mal tumo r s (PN E T s ) (ES ), alveolar rhab- tive R NA-bind i ng d om a in (exo ns 11 ± 1 3 ) d e ® n e d b y do m y osarcoma (ARMS ), ma lign ant m e l anoma of a co ns er ved 8 0 - aa dom a in. Wild- type E W S has soft parts (MM S P o r clea r cel l s arcoma ), de s mo- bee n s hown t o b ind R NA in vitro and E W S/G AL4 plastic s mall round c ell t um o r (DS RCT) , synovial fusion proteins ca n activate a rep orter g en e , s ugg e st- 9,21,23 sarcoma (SS ), and l iposarcoma (LS ), and t he p oten - ing a role f or EW S in t rans cription . tial f or targe ting t he r es ulting c hime r ic p rotein s in FL I 1, a me mb er of the E T S family of trans crip- no v el i mm u no t he rapies . tion f actors, is the h uma n homo l ogu e o f the m u rine FLI1 ge ne a nd i s nor mally exp ressed i n h ema topoi- et ic t issues. Th e E T S DN A -bind i ng d o m a in, us u- Tu m o r-associ ated ch r omo s oma l transloca- ally loca ted i n t he C -ter mina l portion o f the p rotein, tion s i n p ed iatric s arcoma s is an 85 - aa regi on t hat r eco gn i zes targe t g en e s throug h a co ns erved G GAA/T s equen ce . In FLI1, Ew i ng’ s s arcoma/ p r imitive n euroectode rmal tumors the E T S do m a in is enc od e d i n t he C -termi nu s , and Th e E S /P N E T f amily of tum o r s is a gr oup of poorly the N - termi na l regi on c ont ains a dom a in that i s 9,27 di fferen t iated m a ligna ncies that i nc lud e E w ing’ s sar- func tion al in r epor ter ge n e a ssays. co ma (ES ), per ipheral ne ur oep ithel iom a (PN E T ) EW S/F L I 1 i s a poten t t rans cr iption f actor tha t and A skin’ s tum o r . Th ey are thou gh t t o o r iginat e ca n trans form NI H 3T 3 c e lls, and s tudi es h ave from the n e u roec tod e rm, and s how varying, b ut shown t hat s eque nc es in b oth E W S and F L I 1 a re 7± 9 limi ted d eg r ee s of ne ur al di fferentiation . Th ese t u- es sential f or transformation. To b etter de ® n e t he mo r s exp res s MI C2, a me mb rane p rotein t hat a p- func tion al reg ions of the f usion protei n, substitu- pears to f unct ion i n c ellul ar adhes ion. Th e tion s were m a de i n w hich do ma in 1 o f EW S was ex p ression o f this antige n d i sting ui shes thes e tum o r s replaced w ith a strong h et erolog o us act ivation d o - 12,13 from other small roun d c e ll m a lign a ncies . In ma in. Ma ny of thes e fusion proteins retaine d a c- 7,23 addi tion , approxi ma tely 8 5 % of ES /P N E T t um o r s tivity, althoug h no t a ll were t rans forming . 14± 17 are character ized b y t(11 ; 22 ) (q24 ; q12 ). Delat- Do m a in 2 o f EW S co ul d a lso be e xc ha nge d w ith a tre et al. dem o ns trated t hat t he t (11 ;22) (q2 4; q12) wea k trans cr iptiona l activation d o ma in from TL S / rearrang e s the FLI1 ge ne ( Fr iend l euk em i a int e- FU S witho ut l os s of act ivity. Th us, the se d a ta sup- gr ation s ite 1 ) o n c h romosome 1 1 q 24 w ith a hereto- port a mo d el w herein t he E W S regi on o f EW S/F L I 1 8,18 fore u n c ha racter ize d g e n e, EWS. There is no co nf ers strong t ransactivati on t hroug h do m a in 1 ev idenc e f or the e x pr es sion of the r ec ipr ocal h ybrid with a ddi tion a l properties (protei n± protein i nt erac- transcr ipt. tion ) c on tribut ed b y do ma in 2. Mo lecular alterati ons in pedi at ric sarcomas 79 Several v ariants of the t (11 ; 22 ) (q24 ; q12) E W S/ ce lls e x pr es sing E W S/F L I 1 a nd f ound t hat e x - 8,19 FL I 1 g e ne f usion h ave been d e s cr ibed, but most pression of the f usion p rotein a ntisen s e RNA in- 2,28 include EWS exo ns 1± 7 a nd FLI 1 ex on s 8 a nd 9 . cr ea sed suscep tibility t o a poptos is. Th us, Th er ef ore, the a mino t er minal portion o f EW S is EW S/F L I 1 m a y con t ribute t o m a ligna nt t rans form- always fused t o t he c arboxy ter mina l reg ion o f ation b y alter ation o f mo r e t ha n on e g e ne o r ge ne 8,19 FLI1 which sugg es ts that t he se E W S/F L I 1 v ari- pathways. ants con t ribute t o o n co ge n es is by similar me c ha - The ge ne i s also inv olved i n s ev eral other EWS nisms. tum o r-associ ated t rans location s. Fo r exa mple, a EW S/F L I 1 a nd F L I 1 h ave similar DN A -bindi ng mi no r ity of PN E T s pres en t w ith a variant t (21 ; 22 ) 9,29 3,28,36 speci® c i ty a nd a f® ni ties, but E W S/F L I 1 i s a mo r e trans loca tion t hat f uses to the gene. EWS ERG 9,29,30 potent t rans activator than FL I 1. studies Li ke F L I 1, ER G is a me mb er o f the E T S family of In vitro sugge s ted t ha t E W S/F L I 1 f unc tion ed a s a trans acti- trans cription f actors and m a y reg u late s imilar targe t 29 32 vator at 1 0 - fold lowe r co nc e nt ration s than FL I 1. genes. Studi es are und e rwa y to i de nt ify ER G Th us, it i s likel y that E W S/F L I 1 m e di ates i ts trans - targe t g e ne s . Several l ine s of ev iden c e s ugge s t EW S/ forming e f fects, at l east i n p art, by trans activation o f ER G ma y con t ribute t o n e o plastic t rans formation FL I 1 t arge ts o r promo t ers c on taini ng E T S -bindi ng by the s ame o r similar me ch anisms as EW S/F L I 1. sites. Be c aus e c-MYC is upreg u lated i n s ome t u- Fi rst, PN E T s cont aini ng E W S/F L I 1 o r EW S/E R G mo r s, inc l ud ing E S , one p oten tial t arge t g e n e o f are pheno t ypically and c l ini ca lly indi sting u ish- 2,36 EW S/F L I 1 w as thou gh t t o b e c-MYC. A study by able. As is see n i n E W S/F L I 1, EW S/E R G fusions Bailly et al. inv estiga ted t ransactivati on o f c-MYC by include EWS ex o ns 1± 7, with ERG seq uenc es en- 3,28,36 EW S/F L I 1 u s ing t rans ien t t ranf ect ion H eL a cel ls. co di ng t he E T S dom a in. Th e f usion protein Th es e exp erime n ts s ugg es ted t hat E W S/F L I 1 also func tion s as a transcr iption f actor and r eq uires played a role i n i nc reased e x pr es sion of c-MYC. the s ame r eg ions for transactivati on d e ® ne d i n E W S/ Howe ver, d irec t b indi ng o f EW S/F L I 1 t o E T S - FL I 1 s tud i es . Fu r thermo re, c e lls e x p ressing E W S/ bind ing s ites in t he c-MYC promo ter c ou l d n o t b e ER G have a de cr eased a bility to u nd e rgo a poptosis. de t ec ted u sing g el s hift mo bility a ssays. Th us, EW S/ Th ese c e lls c o ul d b e m a de s usce pt ible t o a poptosis FL I 1 u p regu lates , albeit b y an ind i rect by the e xp ression o f EW S/E R G antisen se R NA . c-MYC me c ha nism yet t o b e e luci da ted . Th erefo re, it i s likel y tha t E W S/E R G fusions con- Rece nt s tud i es s ugg es t that E W S/F L I and F L I 1 tribut e t o o nc og e ne sis in a ma nne r similar to E W S/ ex h ibit some d i fference s in D N A -bindi ng a nd p ro- FLI1. tei n± protein i nter actions . Th er ef ore, it i s possible A rare, third v ariant, t (7: 22 ) (p22 ; q12) h as been tha t E W S/F L I 1 a lso co nt ribut es to t rans formation described in which EWS is fused to ETVI, the by activating g en e s no t n o rmally regu l ated b y FL I 1. huma n homo log o f the m u r ine E T S ge ne ER81. It is Stud ies a re on g oi ng t o i de nt ify the n or mal targe ts o f likely that E W S/E T V 1 c o nt ribut es to m a lign a nt EW S/F L I 1 a nd F L I 1. Br aun et al. utilized r epre- trans formation b y me di ating a berrant t rans cription sent ationa l di fference a nalysis (RDA ) t o i de nt ify and/ o r rep ressing e xp ression o f regu l atory ge n es . di fferen t ially ex p ressed gen e s from NI H 3T 3 c el ls Howe v er, RDA analysis of EW S/E T V 1 r ev ealed co nt aini ng E W S/F L I 1 o r nor mal FL I 1. Th is ap- that o n l y on e o f eight E W S/F L I 1 t arge t g en e s was proach reveale d t hat s ev eral transcr ipts were d ep en - up regu l ated b y EW S/E T V 1 . Th is sugge sts tha t de n t o n t he f usion p rotei n fo r ex pr es sion, while a t EW S/E T V 1 a ctivates o nl y a portion o f the E W S/ least t wo t rans cripts wer e r epres sed. Strom e lysin 1, FL I 1 t rans formation p athway, req uiring o t he r a lter - cy toker atin 1 5 , and a mu r ine h omol og o f cy - ation s for tum o r igen e sis, or tha t E W S/E T V 1 p lays a toc hrom e P - 450 F 1 a re all ind u c ed fo llow ing e x - mi no r role i n t rans formation. Fu r ther stud i es are pres sion of EW S/F L I 1. How ever , the k ine t ics o f ne ed e d t o d e ® n e t he e ffect o f EW S/E T V 1 o n n o r - ex p ression a rgue a gains t the d i rec t u p regu l ation o f ma l ge n e e x pr es sion. all of thes e targe t g e ne s. Th e e l uc ida tion o f such Recen t ly, Pe t er et al. ide nt i® e d a ne w m e m b er of primary target s will p rovide i ns igh t i nt o t he r ole o f the E T S family fused t o in E w ing ’ s sarcoma , EWS EW S/F L I 1 i n t rans formation . It i s likely that t he the gene. which ma ps to c hr omo some FEV FEV, on c og en i c p roperties o f EW S/F L I 1 r esults from 2, enc o de s a 23 8 - aa protei n. Its exp ression i s high ly both t he i nappropriate e xp ression o f FL I 1 t arget restrict ed w ith p rotein b eing d e tec ted o n l y in a dult ge n es , as well a s no vel p rotein ± p rotei n i nt er actions prostate a nd s mall int estine s , but n ot i n o t her f etal which ma y lea d to t he a ctivation o f no n - FLI 1 t arget or adult t issues. FE V c on t ains an ET S DN A bind- ge n es . Studi es that u tilized a ntisen se E W S/F L I 1 ing d o ma in closely related t o t ha t o f ER G and cD N A to d i mi ni sh EW S/F L I 1 R NA level s de mo n - FL I 1; howev er , in c on trast t o t hes e proteins , FE V strated m a rkedl y de cr ea sed ce ll g r owth i n v itro, has a small N- ter minal regi on o f on ly 42 a a which the reby i mp lica ting t he f usion p rotein a s a key con - sugg e sts that i t l ack s imp ortant t rans cription r eg ul a- 33,34 tribut or t o a berrant g r owth. EW S/F L I 1 m a y tor y do ma ins present i n o ther ET S family protei ns . co nt ribut e t o o nc o ge ne s is is by inhibition o r alter - It i s unc l ear whether or not E W S/F E V a lters tran- ation o f no r mal apoptot ic p athways. Yi et al. ob- scription o f similar targe t g en e s than other EW S served s uppression o f apoptosis in E w ing ’ s sarcoma fusion protei ns . Fu rther s tud i es are ne ed e d t o 80 T. J. Goletz et al. el uc idate t his fusion p rotei n’ s role i n t he p athoge ne - les s tha n wild-type P A X3, the f usion protein i s a 46± 49 sis of ES. mo r e p oten t t rans activator. Ov erexp ression o f Th e c o mm o n d e no m i na tor of these t um o rs is that mu r ine P A X3 t rans forms NI H 3T 3 c e lls and the all are primitive n e ur oect ode rma l sarcoma s occ ur - PA X3/ F K HR fusion p rotein t rans formed c hicken ring i n c hi ldr en a nd y oung a dults, and t he e v iden c e em b ryo ® broblasts. On e p ossible me ch anism of strongl y imp licate s EW S fusions as key me di ator s of trans formation i s throug h a ga in of funct ion, no t ma ligna nt t ransformation. Th ere i s also strong e vi- on ly by inc r ea sed t rans activation p oten c y, but a lso 49,50 de n ce t o s ugge s t that t he se f usion protei ns con t rib- throug h co ns titut ive a nd i nc reased e xp ression. ut e t o o nc og e ne sis by aberrant e x pr es sion of target Int er es ting l y, a rece nt s tudy which ut ilized a ntisen s e ge n es (activa tion a nd r ep ression) , as wel l a s altering tech nol ogy to d o wn r egu l ate P A X3/ F K HR in A RMS the e x pr es sion of ge ne s no t n o r mally reg ul ated b y tum o r ce lls d e mo ns trated r ed uc e d c ell v iability, the n a tive t ranscr iption f actors. Fu rther more, which l ed t o t he c o nc lus ion t hat P A X3/ F K HR ma y the se g e ne s ma y ef fect n or mal grow th r egu l ation b y co nt ribut e t o m a ligna nt t rans formation t hr ough inter fering w ith a poptotic p athways. suppression o f apoptotic p roce sses which w ou ld no rmally ca use cel l d e ath. Int eresti ng ly, 10 ± 20 % of ARMS tum o rs con tain a Alveolar rha bd o myosarcoma ( ARMS ) variant t rans loca tion, t(1; 13 ) (p36 ; q14) , that r es ults Rhabdom y osarcoma is the m o st c o mm o n s oft tissue in t he i n- frame f usion o f 5 9 PAX7 to 39 FKHR. sarcoma in p ed iatric p atient s, with a pproxima tely PA X7 a nd P A X3 a re highl y homo logo us in t he P B 25 0 c a ses per yea r in t he U ni ted S tates. Rou gh ly and H B d o m a ins, sugg es ting t hat t he y m i gh t r ec og - 40± 43,52 20 % of the se c a ses are of the a lveolar mo r phologi cal ni ze s imilar target g e ne s. Fu r ther mor e, the type ( ARMS ) w hich is ch aracter ized b y alveolar-like PA X3/ F K HR and P A X7/ F K HR chi me ric p rotei ns spaces formed b y ® brovascu lar septa. Th es e spaces share struc tur al similarities in t ha t t hey both c on t ain are ® lled w ith m a ligna nt c el ls that a re disting u ished int act N - ter minal PB a nd H B r eg i ons fused to t he by the ir e os inop hilic cy top lasm. Approxima tely 8 0 % aci di c a nd p roline - rich C-termi na l reg i on o f 41,42,52 of ARMS exp ress a trans location i nv olving t he l ong FKHR. Th erefore , i t i s likely t ha t t hes e arms of ch romosomes 2 a nd 1 3 t (2; 13 ) (q35 ; q14 ) , trans loca tions create s imilar ch imer ic t rans cription which resul ts in t he j uxt apositioni ng o f a trunc a ted factors that c on t ribute t o t rans formation b y altering 50± 52 ge ne o f chr omo some 2 t o t he 3 -terminal ex pr es sion of a com m o n g roup of targe t g e n es . PAX3 9 39± 43 regi on o f the ge ne o f ch romosome 1 3 . FKHR Th e P A X family of trans cr iption f actors play im- Ma lignant melanoma o f soft p ar ts (MMS P ) o r clear cell portant r oles du r ing e m b ryoni c d e v elop ment , p artic- sar coma ( CCS ) ul arly in m o rphoge ne sis and p atter n f ormation . Th es e ge n es co nt ain a paired-box (PB ) D N A - Ma lign a nt m e l anoma of soft parts (MM S P) , also bind ing d o ma in and s ome a lso con t ain a home o box kno w n a s cl ear ce ll s arcoma (CC S), is a rare, but (HB) D N A -binding d o ma in. Ov er ex p ression o f agg r es sive soft tissue s arcoma of mu s cl e t en do n s and the se g e ne s ca n resul t i n o n co ge ni c t rans form- apone ur oses that o c cu rs mo st f requen t ly i n y oung 10,11 53 ation and l oss of func tion m u t ation s has been adul ts betwe e n t he a ge s of 15 a nd 3 5 y ea rs. Over ob served i n s ever al gen e tic d i sea ses, inc lud i ng 95 % of MM S P c a ses oc cu r in t he e x tremi ties , and Waardenb urg s yndr ome . on ly rarely (les s than 2% ) o c cu r in t he h ea d and FK HR, formally know n a s ALV , is a me m b er of ne ck reg i on. Althou gh MM S P i s a me l anin- produc - the f ork-head dom a in (FD ) f amily of trans cription ing t um o r , there i s no e v idenc e t o s ugge s t tha t t hes e factor s which c on tain a co ns er ved D N A -bindi ng tum o rs are direc tly r el ated t o m a lign a nt m e lano m a . mo t if related t o t he Drosophila reg ion- speci ® c h ome - MM S P i s thoug h t t o h ave ne ur oect ode rma l or igi ns otic gene fork-head. Th is family of trans cription and e xp resses ne ur al antige n s, as well a s ma rkers of factor s nor mally func tion s du ring e m b ryoge ne sis. me l anin p roduc tion , such a s HMB - 45. A t(12 ;22 ) The ge n e i s ub iquitou s ly ex pr es sed and fu nc - (q13 ; q12 ) t rans location e ven t i s presen t i n m o re FKHR 55,56 tion s as a trans cr iption fa ctor. than 70 % of these t umo r s and m o lecul ar analy- Th e h ybrid gen e w hich resul ts from the sis of the b reakpoint r evea ls an / fusion. EWS ATF1 t(2; 13 ) (q35 ; q14) t ransloc ation e n co de s a fusion Th is ch imer ic p rotei n j oins the 5 RNA-bind i ng protein c on taini ng t he a mino t er mina l portion o f the regi on o f the ge ne a nd t he 3 regi on o f the EWS 9 PA X3 p rotein i ncl ud i ng t he P B a nd H B d o ma ins ATF1 gen e , a me mb er of the C REB / t rans cr iption joine d t o t he c arboxyl r eg ion o f the F K HR protei n factor family of leuc ine z i pper trans cr iption f actors tha t i s trun c ated w ithin t he w ing e d h elix D N A - that h as a bZIP d o m a in for DN A bindi ng a nd bind ing r egi on , but r et ains a put ative t rans activation protei n± protein i nt eracti on . Th is family of tran- do m a in. Ev iden c e s ugg e sts that t he D N A -bindi ng scription f actor s me d i ates t ranscr iption t hr ough speci® c t y o f PA X3/ F K HR is co nt ribut ed b y PA X3, ATF - bindi ng s ites . Th e e xp ression o f these g e ne s is mo s t likel y through the P B a nd H B d o ma ins, while ind u c ed b y cAMP , and t hey a re act ivated b y phos- FK HR con t ributes the t ransactivati on r egi on . Al- phorylation b y cAMP - dep end e nt p rotein k ina se A 58,59 tho ug h the D N A -binding a ct ivity o f PA X3/ F K HR is (PKA). Mo lecular alterati ons in pedi at ric sarcomas 81 Th e t (12 ; 22 ) t rans loca tion fu ses the N - ter minal Sy novial sar coma ( SS) portion of EWS to t he C -ter mina l regi on o f ATF1, Synovial sarcoma is an agg r es sive soft-tissue m a lig- retaini ng t he b ZIP d o ma in. How ever , the P K A reg - na ncy w hich oc cu r s primarily in t he e xt rem i ties n ea r ul ator y phosphorylation s ite i s los t. Thus, it is ma jor joint s (e. g. a nkle, kne e) o f adoles ce nt s and likel y tha t E W S/A TF 1 c o ul d e xh ibit the D N A -bind - young a dul ts. Vi rtually all synovial sarcoma s con t ain ing s peci® c i ty o f ATF 1 , and d ime r ize w ith C REB , 68 a trans loca tion o f chr omo somes X and 1 8 with but w oul d n o t b e c AMP - induc i ble. EW S/A TF 1 approxima tel y 70 % inv olving t (X; 18 ) (p11 . 2; q11. 2) . do e s act ivate p romo ters w ith A TF 1 b indi ng s ites, Th is trans loca tion e v ent g e n er ates a fusion p rotein althoug h no t a ll such promo ter s wer e a ct ivated , from the 5 reg ion o f the ge ne a nd t he 3 9 SYT 9 and s ome p romo t ers w ere f ound t o b e r epres sed by 69± 71 region of or . Th er e i s no e vide n ce SSX1 SSX2 EW S/A TF 1 . Th erefore, EW S/A TF 1 m a y con t ribute of a trans cript b ei ng e xp res sed b y the r ecipri cal to m a ligna nt t rans formation b y several m e ch anisms. hybrid d e r ( 18 ) . Th e f unct ion o f the SYT gene is Fi rst, EW S/A TF 1 m a y co ns titut ivel y activate A TF 1 un k no wn , and s equen c e a nalysis revea ls no c l assical target g e n es that a re no rmally ind uc e d b y cA MP , or struc tur al mo tifs associated w ith D N A -bindi ng o r it m a y rep ress ge ne s that n or mally func tion i n trans cription a l regu l ation. How ever , the p resenc e o f gr owth c on trol. A lternat ivel y, EW S/A TF1 m a y acti- SH2 a nd S H3 d o ma ins sugg es ts that S YT mi ght vate n o v el g e ne s, perhaps gen e s regu l ated b y othe r func tion t hroug h protei n± protei n i nt eract ion. Th e CREB / A TF f amily me mb er s. rece nt i solation o f the m o u se h omo log o f SYT r e- In m o st MM S P t um o rs, two h ybrid transcr ipts vea led t ha t S YT i s ex pr es sed ubiquitous ly dur ing are gen e rated a nd e xp ressed b y the t (12 ; 22 ) ea rly emb ryogen es is, but e xp ression is res tricted (p13 ;q12 ) t ransloc ation. Th e e x pr es sion pro® le o f later in d e velopm e n t t o c a rtilage t issue, s peci® c n e u- the f usion gen e o n d e r (12 ) c h romos ome i s com p at- rona l ce lls a nd s ome e pithe lial-derived t issues . SYT ible w ith t he u b iquitous exp ression o f ATF . How- was also de tec table i n p rimary sperma toc ytes. ev er, this out -of-frame fu sion resul ts in a produ ct Sev eral studi es sugg es ted t hat S S con taine d t wo co ns isting o f the ® rst 65 N - ter minal amino a ci ds of di stinc t X chromo s ome b reakpoi nt s ites. How ever , ATF 1 , which is un l ikel y to b ind D N A or di me rize, the i den t i® c ation o f two c l os ely related g e ne s at ma king i ts role i n t rans formation u nc l ear. It i s un - Xp11 . 2 e stablished the i nv olveme n t o f di stinc t c od- likel y that e x pr es sion of the d e r(12 ) t rans cr ipt is ing r eg ions . De spite b ei ng 2 M b apart, and SSX1 es sential i n t rans formation g i ven r ep orts that 3 0 % of share 80 % homo logy . Bo t h e nc od e a 18 8 -aa SSX2 MM S P l ack ex p ression. protei n w ith a n N- ter mina l Krup pel-associated b ox (KRAB) t ha t i s thoug h t t o f unc tion a s a trans crip- 72,73 tion r ep ressor dom a in. Althoug h the se p rotei ns Desmo plas tic s mal l round cell t umor (DSR CT ) lack zinc ® ng e r m o tifs, the p resenc e o f the K RAB De s mop lastic small roun d c el l t umo r (DS RCT) i s sequen ce s sugg e st a role i n t rans cr iption. How ever , an agg r essive small roun d c e ll t um o r that o c cu r s this do ma in is not p resent i n t he c h imeric p rotein, pred om i na ntly i n a bdo mi na l ser osal surfaces and which s ugg es ts tha t S SX1 a nd S SX2 s eq uenc e s has a predi lec tion f or youn g m a les. Th e t um o r is a co nt ribut e t o t ransformation t hroug h no vel p rotei n± primitive s mall roun d c e ll w ith f eatur es of di verge nt protei n i nter action s or some o ther funct ion. SSX3, di fferen t iation, co -expr es sing e pithe lial, ne ur al and another KRAB p rotei n, is no t i mp lica ted i n t (X; my ogen i c m a rkers. Th e o rigi n o f this tumo r rema ins 18 ) -positive S S, but h as high h omo logy to S SX1 un c lear, b ut i t i s mo s t likely de rived f rom the and S SX2 ( 95 a nd 9 0 % , respectivel y). Th e s tud y of me s othelium . Almo st 1 0 0 % of these t umo r s co nt ain this ge ne m a y provide i ns igh t i nt o t he f unct ion o f a t(11 ; 22 ) (p13 ; q12) t rans loca tion t hat f uses the 5 9 SSX1 a nd S SX2. regi on o f the EWS gene to the 39 region of WT1, a tum o r suppres sor gen e i nv olved i n a subset o f 62± 66 Lipo sar comas ( LPS) Wilms ’ tum o rs. WT1 b inds DN A throug h a series of zinc ® ng e rs and r epresses the t rans cription Li posarco ma s (LS ) a re soft tissue t um o rs that o cc ur of ce rtain g e ne s. Th es e zinc ® nge r s are es sential f or primarily in t he e xt rem i ties a nd r et roperitone u m. transcr iption a l repres sion. Th e c hi me ric p rotei n Th ese t um o rs are from primitive m e s enc h ymal cells co nt ains the N - ter mina l reg ion o f EW S fused t o t he and t hey res emb le f etal adipose tissue. S ever al WT1 D N A -bindi ng d o m a in. Given t hat b oth the ch aracteristic c y tog e ne tic a berration s have been wild-type EWS ge ne a nd E W S fusion protei ns are ide n ti® e d f or adipose tumo r s. Th e m o st c o mm o n know n t o p artici pate i n t ranscr iption a l co mp lexe s, it LS are my xo id r oun d c el l l iposarcoma s, and g r ea ter is likely t hat E W S/W T1 fu nc tions as a trans cription than 90 % of my xo id l iposarcoma s co nt ain t he factor , possibly throug h WT1 t arget s. Th erefore , t(12 ; 16 ) (q13 ; p11) t rans loca tion i n w hich CHOP un l ike t he l os s of funct ion m u t ation i n W ilm’ s tu- on t he l ong a rm of chromo s ome 1 2 i s fused t o 22,75± 77 mo r , the l os s of the z inc ® nge r regi on o f WT1 i n FUS/TLS. Howe v er, this transloc ation e ven t EW S/W T1 s erves to c o nv ert W T1 f rom a repres sor has no t b een d et ect ed i n o t he r a dipose tum o r s and, of transcr iption t o a do mi nant t ranscr iption a l act iva- ther ef ore, ma y provide i nt eresti ng i ns ight i nt o t he tor on c og en e . trans formation p roce ss of thi s subset o f tum o rs. 82 T. J. Goletz et al. FU S/T L S is struc t ur ally similar to E W S ( . 50% and r ecog n ition h as incr ea sed c on side rably in t he amino a cid i de n tity) and i s exp ressed a t h igh lev el s last two d e ca des and h as bee n e xp er tly review ed 22 81 in a ll tissues exa mine d. TL S binds RNA and elsewhere. Br ie¯ y , T c el ls recog n i ze a ntige n s as en c od es a strong t rans cr iptiona l activation d om a in short pep tide s tha t a re boun d t o t he c e ll s urface i n in t he N - termi na l reg ion. Th er ef ore, like E W S, the c on t ex t o f ma jor histocom p atibility ( MH C) 81,82 1 FU S/T L S ma y funct ion a s a nu c lea r RNA -bindi ng molecules. In t he c a se of CD8 CTL , the T c el l protein. rece ptor (TC R) rec og n izes s hort p ep tide s (8± 10 CHOP , also ca lled G ADD 1 5 3 , is a me m b er of amino a cids) b oun d t o M H C class I mo lec ul es . the C CATT / e n h ance r-bindi ng p rotei n ( C/ E B P ) Th ese p eptide s are de rived f rom end o ge no u s ly ex - family of leu c ine z ipper t rans cr iption f actors that pressed p rotei ns which un de r go p roteol ytic p roce ss- regu l ate a dipocy te d i fferentiation. CHOP i s ex - ing i n t he c ytosol b y large p roteos ome c om p lex es . pres sed at l ow level s in a dipocytes; h owev er, mR NA Pe ptide f ragmen ts a re the n t rans ported i nt o t he levels i nc rease d u r ing c on di tion s of stres s such as lum e n o f the e nd o plasmic r eticul um (ER ) b y spe- DN A da mage . O v erexp ression o f CHOP i n N I H ci alized t rans porters o f antige n p roces sing ( TA P) . 3T 3 c el ls res ults in g r owth a rrest at G 1/ S . Thus, On c e i ns ide t he E R , pep tide s associate w ith a n CHOP i s thou gh t t o fu nc tion a s a do m i na nt n eg a - appropriate M H C cl ass I mo l ec ul e t ha t i s associated tive g r owth r egu l ator. with b eta-2- micr oglobul in ( b 2 m ) , an inv ariant s ub- In t he T L S /C HOP f usion p rotei n, the N - ter minal un i t w hich is thoug h t t o e n h ance e f ® ci ent M H C portion o f TL S is joined t o t he e nt ire C HOP c o di ng foldi ng , o p timi ze M H C/p ep tide b indi ng, and i n- 75,76 region. TL S /C HOP c an t rans form NI H 3T 3 cr ea se stability o f the M H C/p ep tide c o mp lex d ur ing ce lls a nd s tud i es i nd i ca te t hat t rans formation r e- trans port to a nd e xp ression o n t he c el l s urface. quires seq uenc e s from both T L S and C HOP . The Fo l low ing p eptide / M H C bind ing, the p eptide/ requirem e nt f or the C -termi na l leuc ine z ipper do - MHC/b 2 m co mp lexe s transverse t he E R and G olgi ma in of CHOP f or trans formation s ugg e sts a cruc ial apparatus, and a re di splayed o n t he c el l’ s surface role f or C/E B P p rotei n d ime rizati on. Althou gh it i s where t hey are subject t o s urveillance b y CTL . In un c lear w hether nor mal wild- type C HOP a ctivation the c ase o f CD4 Th cells, t he T C R reco gn i ze requires DN A -binding , the p oten t ial D N A -bindi ng sligh tly larger pep tide s (10 ± 25 a a) in t he c on tex t o f regi on , a basic reg ion o f the b ZIP d o m a in, is re- MH C class II mo lec ul es . Th ese p ep tide s are typi- quired f or trans formation. Th e r ole o f TL S se- ca lly de r ived f rom ma terial o r or ga nisms which have quen c es in t rans formation m a y be m o re t han that o f un d e rgon e e n d o/ p hagocy tos is by APC . Th us, in a strong t rans activator , since s ubstitut ion o f this ge ne r al, CD8 CTL rec ogn i ze i nt race llular ( en- regi on w ith o ther poten t t rans activating d o ma ins di d do ge n ou s ) peptides while C D4 T c el ls reco gn i ze no t m e di ate t rans formation . How ev er, substitut ions ex ter na l (ex o ge no u s) p rotei n f ragme nt s. with E W S seq uenc es wer e t ransforming . There- CTL ca n di sting u i sh self from no n- self peptide s fore, TL S /C HOP m a y con tribut e t o t rans formation associated w ith M H C cl ass I mo l ecu l es, so tha t by me ch anisms similar to t ho se p reviously di scu ssed ex pr es sion of viral protein s or alter ed c ellul ar in E W S fusion p rotei ns . protei ns will b e r e¯ e ct ed i n t he p eptide /M H C co m- plexe s displayed o n t he c e ll s urface. Althoug h the tum o r-speci ® c f usion p roteins de s cribed i n t hi s re- Poten t ial imm u n o t her apeut ic a pproach e s f or view func tion a s nu c lea r trans cr iption f actors, they the t reatmen t of p edi atric s arcom a s are still s ubject t o t he p roteo lytic p roces sing a nd Altho ug h mu l ti-mod ality therapy has imp roved s ur- present ation p athways de s cr ibed. Th er e i s exp er - vival rates for the p edi atric s arcoma s de scribe d i n ime n t al e vide n ce t ha t t um o r-associ ated n u c lea r thi s review , patien ts o ften r el apse, at w hich time protei ns , such a s mu t ant p 53, can i ndu c e i mm u n e 83± 88 responses to m u l ti-age nt c h emo t herapy are brief or responses. . no n -exi sten t . F u r ther mor e, patien ts w ho present Th e i de nt i® c a tion o f TA A and a n incr ea sed with m e t astatic d i sease at d iagn o sis do v er y poorly un d e rstand i ng o f the r eq uireme n ts f or the i nd u c tion in s pite o f agg r es sive mu l ti-moda lity ther apy. Th ere- of ce ll-medi ated i mm u n e r esponses (Ta ble 2) h as fore, efforts are ne e de d t o d e velop n o vel t reatme nt s, led t o a dvances in i mm u n o ther apy. While a num- such a s imm u no t he rapies . Stud i es o v er the p ast ber of TA A have been i de nt i® e d f or sev er al tumo r 90± 93 de c ade h ave provided e vide n ce t hat t rea tme n ts types, it i s un cl ea r whet he r a ll TA A will b e based on t he m a nipulation o f the i mm u n e s ystem ef fective t umo r regr ession a ntige ns . Ide ally, one ca n me di ate r egr ession o f es tablished m e tastat ic wou l d l ike t o i den t ify and t arge t T A A which play a ca ncer. M o re s peci® c a lly, cel l-med iated i mm u n i ty key role i n n e o plastic t rans formation, so that t hey ca n play a cr itical r ole i n t um o r regr ession. ca nno t b e l ost w ithou t l oss of ma lign a ncy . Th e T l ymphocy tes are mo s t often c atego r ized a s tum o r-associ ated t rans loca tions ide nt i® e d f or a 1 1 CD8 cy tot oxi c l ymphocy tes (CT L ) o r CD4 nu m b er of ped iatric s arcoma s such as ES and A R helpe r l ymphocytes (Th ), and b oth t ypes of T c el ls ma y ver y wel l b e s uch a ntige ns , since t hey ge ne r ate are kno wn t o p lay a role i n t um o r reg res sion. Ou r func tion al ch imer ic t rans cr iption f actor s kno wn t o un d e rstand ing o f antige n p roce ssing, pres en tation , co nt ribut e t o a bberrant g en e e xp ression. Mo re Mo lecular alterati ons in pedi at ric sarcomas 83 Table 2. Imm u notherap eutic ap proach es using tumor-as sociated a n tigens Ac tive i mm u n otherap y u s ing i mm u nod omi nant p eptides : alone with a dju v ant s linked t o h elper peptide s Ad m i nistered : in l ipids/l iposom e s pulsed o nt o a ntigen- present ing cells ( APCs) Substit ut ed p eptide s imm u no dom i na nt p eptides w ith a mi no a cid s ubstitu tions t o i ncrease bind ing to M H C Proteins alone with a dju v ant s DNA `na ke d’ DNA e nc odi ng c ancer a nt igens a dmi nistere d u sing gene g un int ramu s cul ar i njection associa ted/ l inked t o l ipids Re comb ina nt v iruses recom b inant v irus es, s uch a s v accinia , f owlpox or a de nov irus, e ncod ing cancer a ntigens , a lone or i n c om b ination w ith g enes e nc od ing cytok ines c os timu l atory m o lecul es o r imm u nos timu l atory fa ctors Re comb ina nt b acteria recom b inant b acteria s uch a s b acillus c alme tte± g uerin ( BC G), Salmonella or Listeria enginee red t o e xpress c anc er antigens alone o r w ith g enes e nc odi ng c ytokin es, c ostimu latory mo lecul es o r other i mm u no stimu latory f actor s Ac tive i mm u n otherap y f ollow ed b y c ytokine s Int erleuk in 2 (IL-2), IL-6, IL-10, IL-15 Passive i mm u not herapy w ith a nt i-tumo r l ymp hocyte s g enerate d in vitro Ge neratio n o f CTL us ing i mm u no domi nant p eptide -pulsed A P Cs Ge neratio n o f Th b y c oincub ation o f APC with a ntigeni c p eptides speci® c a lly, the b rea kpoint ju nc t ions are likely n eo - do es not r eact a ga inst all possible antige ni c d e ter mi- antige ns . Fu r ther , it s hould b e p ossible to a void na nts, ch aracterizati on o f the i mm u no d o mi na nt autoimm u n e r esponses by focus ing o n m i ni ma l pep- pep tide s in t he t um o r reg res sion antige ns will f ur- tide s cor respondi ng t o t he s eq uenc es which span the ther aid in t he d e vel opme n t o f effective t reatme nt s. brea kpoint , s ince t hes e wou ld n o t b e p resen t i n Th e i de nt i® c ation o f TA A and t he c l on ing o f the no r mal ce lls. Th is hypothes is was tested i n a nimal ge ne s which enc o de t hem p rovides num e r ous op - mo d e ls u sing s ynthe tic p eptides co rres pond i ng t o portun i ties for the d ev elop ment o f ca ncer t her apies the b reakpoi nt j unc tion s in E S and A RMS as im- (Ta ble 2) . T h erapies co ul d u t ilize t he T A A protein mu n o ge n s. In t hes e studi es , pep tide -pul sed A PC ei ther a lone o r with a djuvants. Alterna tivel y, the admi ni ster ed i nt raveno us ly, ge ne rated C D8 CTL admi ni stration o f peptides de rived f rom the T A A responses ca pable of lysing p ep tide -pulsed t um o r protei n a dminister ed a lone, with a djuvants or in cells in vitro as well a s tumo r ce lls t rans fected t o co mb inatio n w ith h elpe r p ep tide s , has cer tain a d- ex p ress the f ull-leng t h f usion p rotein. Fu rther more, vantage s in t hat t his approach has bee n d e mo n- the se r esponses were a ble to r ed uc e o r irradicate strated t o g e n er ate T c e ll r es pons es while h aving tumor . Th es e data de m o n s trate t hat t he mi ni ma l risk in t he i nd u ct ion o f un wa nted a nd in vivo ch imer ic f usion p roduc t s res ulting f rom ch romo- potent ially da nge rous autoimm u n e r eact ions . Anti- somal trans loca tions can s erve as neo antige ns . tum o r responses ge ne r ated b y peptide v accina tion Be ca use the t rans loca tion e ven t s are tumo r speci® c , ma y be a ugme n t ed b y ma nipulation o f the r out e/ the rapies targe ting t he r es ulting f usion p roteins mo d e o f admi ni stration. Th e c lon i ng o f ge ne s en- woul d b e h igh ly speci ® c a nd p otent ially les s toxi c. co di ng T A A will fa cilitate t hei r exp ression i n Clini ca l trials are cu r rent ly u nd e rwa y in p atien ts high- ef® ci enc y e xp ression s ystems, such as rec om b i- with E S and A RMS to e valua te t he g en e ration o f na nt v iruses or bacter ia. Th ese v ec tor s ca n be e ng i - anti-tum o r responses using a similar approach . In ne er ed t o e x pr es s the T A A alone o r in c onj unc tion addi tion , stud i es a re on g oi ng t o n o t o nl y ide nt ify with c ytokin e g en e s or ge ne s enc od i ng c os timul a- addi tion al TA A, but a lso to g ain a n un de r standi ng tor y mo l ec ul es . Fu r ther mor e, direc t i njec tion i nt o as to w hich T A A ma y serve as tum o r reje ction mu s cl e o f DN A enc od i ng a ntige ns or the u se o f antige ns . Sinc e i t i s cl ear tha t t he i mm u n e s ystem `ge n e g un s ’ in w hich DN A is attache d t o s mall 84 T. J. Goletz et al. particl es t hat a re me ch anically propelled i nt o c el ls promo t er r egi on f rom one o f the p artner ge n es . is also an ef fective m e thod o f ind u ci ng i mm u ne Howe v er, this me c hani sm has no t b een o bserved i n 95± 100 responses. solid tumo r s, but m a y be r el evant i n h ema topoiet ic Ant i-tumo r res pons es have been g e ne r ated b y in ma lign a ncies . No ne t he les s, it i s likely that e x - vitro sens itizati on o f peripheral blood l ymphocytes pression of hybrid proteins in s olid t um o r s dysregu- (PB L ) t o p ep tide -pul sed A PC or irradiated t um o r lates the t rans cr iption o f key gr owth c o nt rol g e ne s or ce lls. Repea ted i n v itro sens itizati on u s ing i m- pathways, ther eb y promo t ing t umo r igen e sis. mu n o do m i na nt p eptides from me l anoma antige ns While f usion p rotein s are likely t o i nvoke a com b i- pulsed o n t o a utolog o us peripheral blood m o n on u - na tion o f the a forem e nt ione d m e c ha nisms, the r e- cl ea r ce lls i n t he p res en ce o f IL-2 resul ted i n t he du n d anc y o f thei r role i n o nc og e ne sis is no t ewo r thy. ex p ansion o f CTL (10 , 00 0 - fold) o ver a 6- week pe- Th e m u l tiple i nt er ch ange o f func tion a l do ma ins riod. Cel ls ge ne rated b y this approach showed i m- from related g e ne s such a s FLI1 and ERG in mu n e r ea ctivity 50 ± 10 0 t ime s gr ea ter than PNETs, PAX3 and PAX7 in ARMS and SSX1 and co rres pond i ng t um o r in® l trating l ymphocytes SSX2 in S S res ult i n s imilar tum o r pheno t ypes. (TIL) and s peci ® c ally rec og n ized t he a ppropriate Do m a in-swap exp erime n ts i nv olving E W S for TL S imm u n o do m i na nt p eptide a s well a s tum o r ce lls a s in T L S /C HOP s howed t hat s ubstitut ions ca n be me a sured b y lysis and c y tokine r el ea se. Stud ies i n ma de w ith l ittle c hang e i n m o rpholog y. H ow ever , ex p erime n tal a nima l mo d e ls s ugg e st t ha t s peci® c ot her e x p erime n ts i n w hich FL I 1 w as ex ch ange d f or tum o r reco gn i tion a s det ermi ne d b y lysis and c y - CHOP i n f usions with T L S or EW S had an ef fect tok ine s ec retion a ssays cor related h ighl y with in vivo on c e ll m o rphology , such that t he m o rpholog y in anti-tum o r effect s. Th es e co rrel ates h ave also some c a ses was de p end e nt o n t he D N A -bind i ng been o bserved i n p atient s treated w ith a utologo us regi on o f the c hime r ic t rans cription fa ctor. F i na lly, 103,104 TIL. In s everal o t her stud ies, T c el ls stimu- of no te i s the e a rly ons et o f ma ny of the se t um o rs. lated in vitro wer e c apable o f recog n i zing a nd l ysing Th is sugg e sts that t he g e ne s inv olved i n s arcoma -as- target c e lls p ulsed w ith p ep tide s know n t o b ind t o a soci ated t ransloc ation s have speci® c p atter ns of de - particu lar MH C cl ass I mo lecu le; h owe v er, thes e vel opme n t al r eg u lation , and t hat d ysregu lation o f same T c el ls were o f ten i nc apable o f recog n izing this temp oral regu l ation h as profoun d e f fects. and l ysing t he l ow levels o f proces sed p ep tide s ex - Attem p ts at d ev elop ing n e w t her apeut ic a p- pres sed by tumo r cel ls. Th us, ther e i s co ns ider - proach es to t he t rea tme n t o f thes e tum o rs have able h eter ogen e ity i n a nti-tum o r responses . inc l ud ed i mm u n o ther apy. Howe ver , succe ssful i m- mu n o ther apeut ic s tratagi es mu st m e e t s ever al cri- teria, t he ® rst of which is the e xp ression o f TA A tha t Summary are rec og ni ze d b y T l ymphocy tes . In t he c a se of the Th e g e ne ration o f chi me ric t rans cr iption f actors is a sarcoma s present ed i n t his review , the c hi me ric co mm o n c on sequen c e o f ch romos oma l trans loca - trans cription fa ctors represent p oten t ial T A A. Stud- tion s in s olid t um o r s. Th e r esulti ng f usion p roteins ies in e x p erime n tal a nimals sugg es t tha t t he t rans lo- have been s hown, in s ev eral ca ses, to h ave trans - ca tion b rea kpoints in E S and A RMS rep resen t forming a ctivity . Chime ric o n co proteins ma y func - ne oa ntigen s which can b e r ec og ni zed b y CTL . Fu r - tion t hroug h sev er al me ch anisms. Fi rst, a strong ther mor e, these r esponse w er e s uf® cient t o m e di ate act ivation d o ma in from one g e ne m a y be f used to in vivo tum o r reg ression i n a nima l mo de l s. Clini cal the D N A -bindi ng s peci ® c ity r egi on o f another ge ne , vacci ne s tud i es are on g oi ng t o e v aluate t he a bility of leadi ng t o d y sregu l ated e xp ression o f targe t g en e s. thes e TA A to s erve as tum o r regr ession a ntige ns . Th e f usion p rotei ns associated w ith M M S P, ARMS , Fi nally, iden t i® c ation o f the i mm u n o do mi na nt e p i- and P N E T s are exa mples of this me cha nism. How- top es in t um o r reg r es sion antige ns will f avor the ev er, in m y xo id l iposarcoma , the F U S/C HOP g e ne ind u c tion o f ef fective a nti-tu mo r responses. Scr een- produ ct a ppears to m e di ate i ts effect o n t ranscr ip- ing v accine s and v arious de livery systems (pept ide s tion t hroug h protein ± p rotei n i nter actions and m a y or proteins in a djuvants or on d en d ritic c e lls, DN A , no t r eq uire D N A -binding. Sec ond , a fusion partne r viruses ) in a nima ls, such as HLA -transgen ics, will ma y con tribut e m o re t han an act ivation d o m a in. hel p to i den t ify the m o st p romising v accine s for us e Fo r exa mple, the E W S/F L I 1 f usion protein o f ES in c l ini ca l trials. seem s to c o mb ine t he t ransactivati on d om a in of EW S with t he D N A -binding r egi on o f FL I 1. How- References ev er, the f usion p rotein a ppears to m e di ate n o vel protein± protein /p rotei n± nu c leic a cid i nt er actions . 1 Ra bbitts T H. Chromo soma l t ransloc ation s i n h um a n Also, the c hi me ric o n c op rotein m a y het er odi me rize cancer. Nature 1994; 372:143± 9. 2 Delattre O , Zucma n J , Me lot T , et al. The ewing with o the r t rans cr iption f actors. Fo r exa mple, the fami ly o f tumo r sÐ a s ub group o f sma ll-roun d-cell heterodi me r ization o f TL S /C HOP w ith C /E B P w ith tumo r s d e ® ned b y s peci® c chime ric trans cripts. New C/E B P f amily me m b ers reg u lates adipocy te g r owth Engl J M ed 1994; 331:294± 9. in a do m i na nt-neg ative m a nne r. F i na lly, chi me ric 3 Gi ov ann ini M , Biegel J A, Serra M, et al. EWS-erg ge n es ma y be o v er exp ressed a s a res ult o f a strong and E WS-Fli1 f usion t ranscri pts i n e wing ’ s s arcoma Mo lecular alterati ons in pedi at ric sarcomas 85 and p rimi tive n e uroe ctode rma l t umo rs w ith v ariant with n ovel r egul ator y d om a ins. Oncogene 1994; transloc ation s. J Cl in Inve st 1994; 94:489± 96. 9:3087± 3097. 4 Downi ng J R, Head D R, P arham D M, et al. Detec- 22 Am a n P , Panagop oulos I , Lassen C , et al. 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Molecular Alterations in Pediatric Sarcomas: Potential Targets for Immunotherapy

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Abstract

Pu rpose/r esults/di scussion. Re cur rent c hromo som a l t rans locatio ns a re com m o n f eatur es o f ma ny h uma n m a lignanc ies. While s uch t ransloc ations o ft en s erve a s d iagnos tic m a rkers, m o lecular analysi s o f these b reakpoi nt r egions a nd t he characte rization o f the a ffected gene s i s l eadi ng t o a g reater und erstand ing o f the c ausal r ole s uch t ransloc ations p lay i n ma lignant t ransfor ma tion. A c omm o n t heme that i s e me rging from t he s tud y o f tum o r-assoc iated t ransloc ation s i s t he generati on o f chime ric gene s t hat, w hen e xpresse d, frequent ly r etain m a ny o f the f unc tional p roperti es o f the w ild-typ e genes f rom w hich they o r iginated . S arcoma s, i n p articul ar, h arbor c hime ric gene s t hat a re often d erived fr om t rans cription factors, suggest ing t hat t he r esul ting chime ric transcri ption fa ctor s c ont ribut e t o t um o rigenes is. T he tumo r-speci ® c expressi on o f the fu s ion p roteins m a ke t hem l ikely c andi dates fo r tum o r-assoc iated a ntigens ( TAA ) a nd a re thus o f int erest in t he d e velopme nt o f ne w therapie s. T he focus o f this r eview will b e on t he t ransloc ation e vents a ssociat ed w ith E wing’ s sarcoma s/P NETs ( ES), a lveol ar r habdom y osarcom a ( AR MS ), m a ligna nt m e lanoma o f soft p arts ( MM S P) (clear c ell sarcoma ), d esmo plastic sma ll r oun d c ell t um o r ( DSRCT ), synovia l s arcoma ( SS) , and l iposarc oma ( LS ), a nd t he p otent ial for targeting the r esul ting c hime ric proteins i n n ov el i mm u n ot herapies . Int roduc tion An a lter na tive c o ns equen ce o f ch romosomal trans loca tions is the g e ne ration o f funct iona l Chromos omal abnormalities are com m o n i n h uma n ch imer ic g e n es . Th is sce na rio is mo st c om m o n i n tum o rs with m a ny ma lign anc ies e xh ibiting c l on al solid tum o rs and u s ually inv olves un rel ated g e n es . ch romos oma l aberrations . Th e i de nt i® c ation o f tu- Of ten, the se t ransloc ation e ven ts a ffect g e ne s en c od- mo r -speci® c c hr omo somal trans location s aids in d i - ing t rans cr iption f actor s, ther eb y ge ne rating 2± 6 agn o sis and s erves as a progn os tic i ndi cator. With ch imer ic t rans cr iption f actors with p roperties of an inc reasing u n d e rstand i ng o f the e f fect t hes e both g e ne s (Ta ble 1 ) . T h e f usion protei ns often ev ent s have on n or mal cel lul ar proce sses, no v el t her - ex hi bit t he D N A -binding s peci ® c ity o f on e g e ne apies c an b e d e velope d w hich have gr ea ter with t he a ctivation d o ma in of the o ther ge ne . S uc h speci® c i ty a nd e f ® ca cy. fusion protei ns act ivate/ r epres s trans cr iption, ex h ibit Tw o m a jor con sequen c es of ch romos omal rear- alter ed D N A bindi ng s peci® c i ty o r partici pate i n rangem e nt s in t um o rs have been i de n ti® e d : the a cti- no vel p rotein± protei n i nt eracti on s . Th us, the y a re vation o f an on c og en e , o r the c reatio n o f a no vel thou g ht t o p lay a cr itical r ole i n t he n e o plastic on c og en i c p rotei n. Fi rst, trans location s can r es ult i n trans formation p roce ss. the a ctivation o f ge ne s located a t o r nea r the b reak- Th e i de nt i® c a tion o f trans loca tions associated point . O f ten, the se g e n es nor mally func tion i n t he with a group of primitive s arcoma s, and t he s ub- promo tion o f cel l g r owth a nd d i fferentiation . Th us, sequen t c lon i ng o f the c h romos oma l breakpoint r e- the ir d isruption c an a ffect n o r mal ce ll r egu l ation . gi on s, has rev ealed t hat a co mm o n t heme i n t hes e Th is type o f alterat ion, which is mo s t com m o n i n tum o rs is the g en e ration o f chi me ric t rans cription hema tolog i cal m a lign a nci es , is illus trated b y the factors. Th e f usion p roteins are ex pr es sed e xc lu- t(8; 14 ) t rans loca tion a ssociated w ith B u rkitt’ s sively in t he t um o r ce lls, and f unc tion a s poten t lymphoma in w hich c-MYC is activated b y repos i- trans cription f actors where t hey a re thou g ht t o c on- tion i ng u n de r the c o nt rol o f the p otent I g e nh ance r. tribut e t o n e o plastic t rans formation b y me d i ating Co rrespon dence t o: L. J . Helman, Mo lecular O n cology S ection, Pediatri c B ranch, Di vision o f C l inical S ciences , National C a ncer I nstitute , Na tional I nstitute s of H ealth, B uildi ng 1 0, Room 13N240, 9000 Rockvill e P ike, B ethesda, M D 2 0892± 1928, USA. Tel: 1 1 301 4964257; Fax: 1 1 301 4020575; E-ma il: h elmanl@p bmac.nci.ni h.gov. 1357-714X/ 9 8/020077± 11 Ó 1998 Ca rfax P ublishing L t d 78 T. J. Goletz et al. Table 1. Tumor-speci® c translocations associated with solid t umors 59 /39 fusion Tumor Transloca tion product Type Ewing’ s s arcoma / 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 rhabdo my osarco ma t(1;13)(p36;q14) PAX7/FKHR Me lano ma o f soft parts t (12;22)(q13;q12) EWS/ATF1 RN A b indi ng/ (clear c ell s arcoma ) bZIP TF DSRCT t(11;22)(p13;q12) EWS/WT1 RN A b indi ng/ Zn ® nger TF Syno vial s arcom a t(X;18)(p11.2;q11.2) SYT /S SX1 SH2 /K R A B b ox SYT/SSX2 Liposar coma t(12:16)(q13;p11) CHOP/FU S -TLS R N A b indi ng/ (my xoid a nd r ound c ell) bZip TF aberrant e xp res sion of no rmal ge n es . Sev eral of the EWS en c od es a 65 6 -aa protei n, the f unct ion o f ch imer ic g e ne s have bee n c lon e d a nd f ound t o c on - which r em a ins un cl ea r. While t his protein i s ubiqui- fer a transformed p hen ot ype w hen e x pr es sed in tou sly exp ressed, exp ression l evel s ¯ u c tua te w ith t he 7± 11 19± 23 vitro. Th e t um o r -speci® c e xp ression o f the f usion cell cycle. EWS con tains two m a jor funct iona l proteins ma ke them likely ca ndi da tes f or tum o r - do ma ins. Th e ® rst is the N - ter mina l regi on ( ex o ns associated a ntige ns (TA A), in w hich the j unc tion 1± 7) c o ns isting o f a ser ies of de ge n er ate r epea ts tha t point c reates a ne o- antige n i c d e termi na nt. T h e f o- resemb le t he t rans activation d o m a ins of sever al cu s of this review will b e o n t he t rans loca tion e ven ts trans cription f actors, such a s SP- 1 while t he s e- associated w ith E w ing ’ s sarcoma s/primitive n e u - co nd r egi on, the C -termi na l regi on , inc lude s a put a- roec tod er mal tumo r s (PN E T s ) (ES ), alveolar rhab- tive R NA-bind i ng d om a in (exo ns 11 ± 1 3 ) d e ® n e d b y do m y osarcoma (ARMS ), ma lign ant m e l anoma of a co ns er ved 8 0 - aa dom a in. Wild- type E W S has soft parts (MM S P o r clea r cel l s arcoma ), de s mo- bee n s hown t o b ind R NA in vitro and E W S/G AL4 plastic s mall round c ell t um o r (DS RCT) , synovial fusion proteins ca n activate a rep orter g en e , s ugg e st- 9,21,23 sarcoma (SS ), and l iposarcoma (LS ), and t he p oten - ing a role f or EW S in t rans cription . tial f or targe ting t he r es ulting c hime r ic p rotein s in FL I 1, a me mb er of the E T S family of trans crip- no v el i mm u no t he rapies . tion f actors, is the h uma n homo l ogu e o f the m u rine FLI1 ge ne a nd i s nor mally exp ressed i n h ema topoi- et ic t issues. Th e E T S DN A -bind i ng d o m a in, us u- Tu m o r-associ ated ch r omo s oma l transloca- ally loca ted i n t he C -ter mina l portion o f the p rotein, tion s i n p ed iatric s arcoma s is an 85 - aa regi on t hat r eco gn i zes targe t g en e s throug h a co ns erved G GAA/T s equen ce . In FLI1, Ew i ng’ s s arcoma/ p r imitive n euroectode rmal tumors the E T S do m a in is enc od e d i n t he C -termi nu s , and Th e E S /P N E T f amily of tum o r s is a gr oup of poorly the N - termi na l regi on c ont ains a dom a in that i s 9,27 di fferen t iated m a ligna ncies that i nc lud e E w ing’ s sar- func tion al in r epor ter ge n e a ssays. co ma (ES ), per ipheral ne ur oep ithel iom a (PN E T ) EW S/F L I 1 i s a poten t t rans cr iption f actor tha t and A skin’ s tum o r . Th ey are thou gh t t o o r iginat e ca n trans form NI H 3T 3 c e lls, and s tudi es h ave from the n e u roec tod e rm, and s how varying, b ut shown t hat s eque nc es in b oth E W S and F L I 1 a re 7± 9 limi ted d eg r ee s of ne ur al di fferentiation . Th ese t u- es sential f or transformation. To b etter de ® n e t he mo r s exp res s MI C2, a me mb rane p rotein t hat a p- func tion al reg ions of the f usion protei n, substitu- pears to f unct ion i n c ellul ar adhes ion. Th e tion s were m a de i n w hich do ma in 1 o f EW S was ex p ression o f this antige n d i sting ui shes thes e tum o r s replaced w ith a strong h et erolog o us act ivation d o - 12,13 from other small roun d c e ll m a lign a ncies . In ma in. Ma ny of thes e fusion proteins retaine d a c- 7,23 addi tion , approxi ma tely 8 5 % of ES /P N E T t um o r s tivity, althoug h no t a ll were t rans forming . 14± 17 are character ized b y t(11 ; 22 ) (q24 ; q12 ). Delat- Do m a in 2 o f EW S co ul d a lso be e xc ha nge d w ith a tre et al. dem o ns trated t hat t he t (11 ;22) (q2 4; q12) wea k trans cr iptiona l activation d o ma in from TL S / rearrang e s the FLI1 ge ne ( Fr iend l euk em i a int e- FU S witho ut l os s of act ivity. Th us, the se d a ta sup- gr ation s ite 1 ) o n c h romosome 1 1 q 24 w ith a hereto- port a mo d el w herein t he E W S regi on o f EW S/F L I 1 8,18 fore u n c ha racter ize d g e n e, EWS. There is no co nf ers strong t ransactivati on t hroug h do m a in 1 ev idenc e f or the e x pr es sion of the r ec ipr ocal h ybrid with a ddi tion a l properties (protei n± protein i nt erac- transcr ipt. tion ) c on tribut ed b y do ma in 2. Mo lecular alterati ons in pedi at ric sarcomas 79 Several v ariants of the t (11 ; 22 ) (q24 ; q12) E W S/ ce lls e x pr es sing E W S/F L I 1 a nd f ound t hat e x - 8,19 FL I 1 g e ne f usion h ave been d e s cr ibed, but most pression of the f usion p rotein a ntisen s e RNA in- 2,28 include EWS exo ns 1± 7 a nd FLI 1 ex on s 8 a nd 9 . cr ea sed suscep tibility t o a poptos is. Th us, Th er ef ore, the a mino t er minal portion o f EW S is EW S/F L I 1 m a y con t ribute t o m a ligna nt t rans form- always fused t o t he c arboxy ter mina l reg ion o f ation b y alter ation o f mo r e t ha n on e g e ne o r ge ne 8,19 FLI1 which sugg es ts that t he se E W S/F L I 1 v ari- pathways. ants con t ribute t o o n co ge n es is by similar me c ha - The ge ne i s also inv olved i n s ev eral other EWS nisms. tum o r-associ ated t rans location s. Fo r exa mple, a EW S/F L I 1 a nd F L I 1 h ave similar DN A -bindi ng mi no r ity of PN E T s pres en t w ith a variant t (21 ; 22 ) 9,29 3,28,36 speci® c i ty a nd a f® ni ties, but E W S/F L I 1 i s a mo r e trans loca tion t hat f uses to the gene. EWS ERG 9,29,30 potent t rans activator than FL I 1. studies Li ke F L I 1, ER G is a me mb er o f the E T S family of In vitro sugge s ted t ha t E W S/F L I 1 f unc tion ed a s a trans acti- trans cription f actors and m a y reg u late s imilar targe t 29 32 vator at 1 0 - fold lowe r co nc e nt ration s than FL I 1. genes. Studi es are und e rwa y to i de nt ify ER G Th us, it i s likel y that E W S/F L I 1 m e di ates i ts trans - targe t g e ne s . Several l ine s of ev iden c e s ugge s t EW S/ forming e f fects, at l east i n p art, by trans activation o f ER G ma y con t ribute t o n e o plastic t rans formation FL I 1 t arge ts o r promo t ers c on taini ng E T S -bindi ng by the s ame o r similar me ch anisms as EW S/F L I 1. sites. Be c aus e c-MYC is upreg u lated i n s ome t u- Fi rst, PN E T s cont aini ng E W S/F L I 1 o r EW S/E R G mo r s, inc l ud ing E S , one p oten tial t arge t g e n e o f are pheno t ypically and c l ini ca lly indi sting u ish- 2,36 EW S/F L I 1 w as thou gh t t o b e c-MYC. A study by able. As is see n i n E W S/F L I 1, EW S/E R G fusions Bailly et al. inv estiga ted t ransactivati on o f c-MYC by include EWS ex o ns 1± 7, with ERG seq uenc es en- 3,28,36 EW S/F L I 1 u s ing t rans ien t t ranf ect ion H eL a cel ls. co di ng t he E T S dom a in. Th e f usion protein Th es e exp erime n ts s ugg es ted t hat E W S/F L I 1 also func tion s as a transcr iption f actor and r eq uires played a role i n i nc reased e x pr es sion of c-MYC. the s ame r eg ions for transactivati on d e ® ne d i n E W S/ Howe ver, d irec t b indi ng o f EW S/F L I 1 t o E T S - FL I 1 s tud i es . Fu r thermo re, c e lls e x p ressing E W S/ bind ing s ites in t he c-MYC promo ter c ou l d n o t b e ER G have a de cr eased a bility to u nd e rgo a poptosis. de t ec ted u sing g el s hift mo bility a ssays. Th us, EW S/ Th ese c e lls c o ul d b e m a de s usce pt ible t o a poptosis FL I 1 u p regu lates , albeit b y an ind i rect by the e xp ression o f EW S/E R G antisen se R NA . c-MYC me c ha nism yet t o b e e luci da ted . Th erefo re, it i s likel y tha t E W S/E R G fusions con- Rece nt s tud i es s ugg es t that E W S/F L I and F L I 1 tribut e t o o nc og e ne sis in a ma nne r similar to E W S/ ex h ibit some d i fference s in D N A -bindi ng a nd p ro- FLI1. tei n± protein i nter actions . Th er ef ore, it i s possible A rare, third v ariant, t (7: 22 ) (p22 ; q12) h as been tha t E W S/F L I 1 a lso co nt ribut es to t rans formation described in which EWS is fused to ETVI, the by activating g en e s no t n o rmally regu l ated b y FL I 1. huma n homo log o f the m u r ine E T S ge ne ER81. It is Stud ies a re on g oi ng t o i de nt ify the n or mal targe ts o f likely that E W S/E T V 1 c o nt ribut es to m a lign a nt EW S/F L I 1 a nd F L I 1. Br aun et al. utilized r epre- trans formation b y me di ating a berrant t rans cription sent ationa l di fference a nalysis (RDA ) t o i de nt ify and/ o r rep ressing e xp ression o f regu l atory ge n es . di fferen t ially ex p ressed gen e s from NI H 3T 3 c el ls Howe v er, RDA analysis of EW S/E T V 1 r ev ealed co nt aini ng E W S/F L I 1 o r nor mal FL I 1. Th is ap- that o n l y on e o f eight E W S/F L I 1 t arge t g en e s was proach reveale d t hat s ev eral transcr ipts were d ep en - up regu l ated b y EW S/E T V 1 . Th is sugge sts tha t de n t o n t he f usion p rotei n fo r ex pr es sion, while a t EW S/E T V 1 a ctivates o nl y a portion o f the E W S/ least t wo t rans cripts wer e r epres sed. Strom e lysin 1, FL I 1 t rans formation p athway, req uiring o t he r a lter - cy toker atin 1 5 , and a mu r ine h omol og o f cy - ation s for tum o r igen e sis, or tha t E W S/E T V 1 p lays a toc hrom e P - 450 F 1 a re all ind u c ed fo llow ing e x - mi no r role i n t rans formation. Fu r ther stud i es are pres sion of EW S/F L I 1. How ever , the k ine t ics o f ne ed e d t o d e ® n e t he e ffect o f EW S/E T V 1 o n n o r - ex p ression a rgue a gains t the d i rec t u p regu l ation o f ma l ge n e e x pr es sion. all of thes e targe t g e ne s. Th e e l uc ida tion o f such Recen t ly, Pe t er et al. ide nt i® e d a ne w m e m b er of primary target s will p rovide i ns igh t i nt o t he r ole o f the E T S family fused t o in E w ing ’ s sarcoma , EWS EW S/F L I 1 i n t rans formation . It i s likely that t he the gene. which ma ps to c hr omo some FEV FEV, on c og en i c p roperties o f EW S/F L I 1 r esults from 2, enc o de s a 23 8 - aa protei n. Its exp ression i s high ly both t he i nappropriate e xp ression o f FL I 1 t arget restrict ed w ith p rotein b eing d e tec ted o n l y in a dult ge n es , as well a s no vel p rotein ± p rotei n i nt er actions prostate a nd s mall int estine s , but n ot i n o t her f etal which ma y lea d to t he a ctivation o f no n - FLI 1 t arget or adult t issues. FE V c on t ains an ET S DN A bind- ge n es . Studi es that u tilized a ntisen se E W S/F L I 1 ing d o ma in closely related t o t ha t o f ER G and cD N A to d i mi ni sh EW S/F L I 1 R NA level s de mo n - FL I 1; howev er , in c on trast t o t hes e proteins , FE V strated m a rkedl y de cr ea sed ce ll g r owth i n v itro, has a small N- ter minal regi on o f on ly 42 a a which the reby i mp lica ting t he f usion p rotein a s a key con - sugg e sts that i t l ack s imp ortant t rans cription r eg ul a- 33,34 tribut or t o a berrant g r owth. EW S/F L I 1 m a y tor y do ma ins present i n o ther ET S family protei ns . co nt ribut e t o o nc o ge ne s is is by inhibition o r alter - It i s unc l ear whether or not E W S/F E V a lters tran- ation o f no r mal apoptot ic p athways. Yi et al. ob- scription o f similar targe t g en e s than other EW S served s uppression o f apoptosis in E w ing ’ s sarcoma fusion protei ns . Fu rther s tud i es are ne ed e d t o 80 T. J. Goletz et al. el uc idate t his fusion p rotei n’ s role i n t he p athoge ne - les s tha n wild-type P A X3, the f usion protein i s a 46± 49 sis of ES. mo r e p oten t t rans activator. Ov erexp ression o f Th e c o mm o n d e no m i na tor of these t um o rs is that mu r ine P A X3 t rans forms NI H 3T 3 c e lls and the all are primitive n e ur oect ode rma l sarcoma s occ ur - PA X3/ F K HR fusion p rotein t rans formed c hicken ring i n c hi ldr en a nd y oung a dults, and t he e v iden c e em b ryo ® broblasts. On e p ossible me ch anism of strongl y imp licate s EW S fusions as key me di ator s of trans formation i s throug h a ga in of funct ion, no t ma ligna nt t ransformation. Th ere i s also strong e vi- on ly by inc r ea sed t rans activation p oten c y, but a lso 49,50 de n ce t o s ugge s t that t he se f usion protei ns con t rib- throug h co ns titut ive a nd i nc reased e xp ression. ut e t o o nc og e ne sis by aberrant e x pr es sion of target Int er es ting l y, a rece nt s tudy which ut ilized a ntisen s e ge n es (activa tion a nd r ep ression) , as wel l a s altering tech nol ogy to d o wn r egu l ate P A X3/ F K HR in A RMS the e x pr es sion of ge ne s no t n o r mally reg ul ated b y tum o r ce lls d e mo ns trated r ed uc e d c ell v iability, the n a tive t ranscr iption f actors. Fu rther more, which l ed t o t he c o nc lus ion t hat P A X3/ F K HR ma y the se g e ne s ma y ef fect n or mal grow th r egu l ation b y co nt ribut e t o m a ligna nt t rans formation t hr ough inter fering w ith a poptotic p athways. suppression o f apoptotic p roce sses which w ou ld no rmally ca use cel l d e ath. Int eresti ng ly, 10 ± 20 % of ARMS tum o rs con tain a Alveolar rha bd o myosarcoma ( ARMS ) variant t rans loca tion, t(1; 13 ) (p36 ; q14) , that r es ults Rhabdom y osarcoma is the m o st c o mm o n s oft tissue in t he i n- frame f usion o f 5 9 PAX7 to 39 FKHR. sarcoma in p ed iatric p atient s, with a pproxima tely PA X7 a nd P A X3 a re highl y homo logo us in t he P B 25 0 c a ses per yea r in t he U ni ted S tates. Rou gh ly and H B d o m a ins, sugg es ting t hat t he y m i gh t r ec og - 40± 43,52 20 % of the se c a ses are of the a lveolar mo r phologi cal ni ze s imilar target g e ne s. Fu r ther mor e, the type ( ARMS ) w hich is ch aracter ized b y alveolar-like PA X3/ F K HR and P A X7/ F K HR chi me ric p rotei ns spaces formed b y ® brovascu lar septa. Th es e spaces share struc tur al similarities in t ha t t hey both c on t ain are ® lled w ith m a ligna nt c el ls that a re disting u ished int act N - ter minal PB a nd H B r eg i ons fused to t he by the ir e os inop hilic cy top lasm. Approxima tely 8 0 % aci di c a nd p roline - rich C-termi na l reg i on o f 41,42,52 of ARMS exp ress a trans location i nv olving t he l ong FKHR. Th erefore , i t i s likely t ha t t hes e arms of ch romosomes 2 a nd 1 3 t (2; 13 ) (q35 ; q14 ) , trans loca tions create s imilar ch imer ic t rans cription which resul ts in t he j uxt apositioni ng o f a trunc a ted factors that c on t ribute t o t rans formation b y altering 50± 52 ge ne o f chr omo some 2 t o t he 3 -terminal ex pr es sion of a com m o n g roup of targe t g e n es . PAX3 9 39± 43 regi on o f the ge ne o f ch romosome 1 3 . FKHR Th e P A X family of trans cr iption f actors play im- Ma lignant melanoma o f soft p ar ts (MMS P ) o r clear cell portant r oles du r ing e m b ryoni c d e v elop ment , p artic- sar coma ( CCS ) ul arly in m o rphoge ne sis and p atter n f ormation . Th es e ge n es co nt ain a paired-box (PB ) D N A - Ma lign a nt m e l anoma of soft parts (MM S P) , also bind ing d o ma in and s ome a lso con t ain a home o box kno w n a s cl ear ce ll s arcoma (CC S), is a rare, but (HB) D N A -binding d o ma in. Ov er ex p ression o f agg r es sive soft tissue s arcoma of mu s cl e t en do n s and the se g e ne s ca n resul t i n o n co ge ni c t rans form- apone ur oses that o c cu rs mo st f requen t ly i n y oung 10,11 53 ation and l oss of func tion m u t ation s has been adul ts betwe e n t he a ge s of 15 a nd 3 5 y ea rs. Over ob served i n s ever al gen e tic d i sea ses, inc lud i ng 95 % of MM S P c a ses oc cu r in t he e x tremi ties , and Waardenb urg s yndr ome . on ly rarely (les s than 2% ) o c cu r in t he h ea d and FK HR, formally know n a s ALV , is a me m b er of ne ck reg i on. Althou gh MM S P i s a me l anin- produc - the f ork-head dom a in (FD ) f amily of trans cription ing t um o r , there i s no e v idenc e t o s ugge s t tha t t hes e factor s which c on tain a co ns er ved D N A -bindi ng tum o rs are direc tly r el ated t o m a lign a nt m e lano m a . mo t if related t o t he Drosophila reg ion- speci ® c h ome - MM S P i s thoug h t t o h ave ne ur oect ode rma l or igi ns otic gene fork-head. Th is family of trans cription and e xp resses ne ur al antige n s, as well a s ma rkers of factor s nor mally func tion s du ring e m b ryoge ne sis. me l anin p roduc tion , such a s HMB - 45. A t(12 ;22 ) The ge n e i s ub iquitou s ly ex pr es sed and fu nc - (q13 ; q12 ) t rans location e ven t i s presen t i n m o re FKHR 55,56 tion s as a trans cr iption fa ctor. than 70 % of these t umo r s and m o lecul ar analy- Th e h ybrid gen e w hich resul ts from the sis of the b reakpoint r evea ls an / fusion. EWS ATF1 t(2; 13 ) (q35 ; q14) t ransloc ation e n co de s a fusion Th is ch imer ic p rotei n j oins the 5 RNA-bind i ng protein c on taini ng t he a mino t er mina l portion o f the regi on o f the ge ne a nd t he 3 regi on o f the EWS 9 PA X3 p rotein i ncl ud i ng t he P B a nd H B d o ma ins ATF1 gen e , a me mb er of the C REB / t rans cr iption joine d t o t he c arboxyl r eg ion o f the F K HR protei n factor family of leuc ine z i pper trans cr iption f actors tha t i s trun c ated w ithin t he w ing e d h elix D N A - that h as a bZIP d o m a in for DN A bindi ng a nd bind ing r egi on , but r et ains a put ative t rans activation protei n± protein i nt eracti on . Th is family of tran- do m a in. Ev iden c e s ugg e sts that t he D N A -bindi ng scription f actor s me d i ates t ranscr iption t hr ough speci® c t y o f PA X3/ F K HR is co nt ribut ed b y PA X3, ATF - bindi ng s ites . Th e e xp ression o f these g e ne s is mo s t likel y through the P B a nd H B d o ma ins, while ind u c ed b y cAMP , and t hey a re act ivated b y phos- FK HR con t ributes the t ransactivati on r egi on . Al- phorylation b y cAMP - dep end e nt p rotein k ina se A 58,59 tho ug h the D N A -binding a ct ivity o f PA X3/ F K HR is (PKA). Mo lecular alterati ons in pedi at ric sarcomas 81 Th e t (12 ; 22 ) t rans loca tion fu ses the N - ter minal Sy novial sar coma ( SS) portion of EWS to t he C -ter mina l regi on o f ATF1, Synovial sarcoma is an agg r es sive soft-tissue m a lig- retaini ng t he b ZIP d o ma in. How ever , the P K A reg - na ncy w hich oc cu r s primarily in t he e xt rem i ties n ea r ul ator y phosphorylation s ite i s los t. Thus, it is ma jor joint s (e. g. a nkle, kne e) o f adoles ce nt s and likel y tha t E W S/A TF 1 c o ul d e xh ibit the D N A -bind - young a dul ts. Vi rtually all synovial sarcoma s con t ain ing s peci® c i ty o f ATF 1 , and d ime r ize w ith C REB , 68 a trans loca tion o f chr omo somes X and 1 8 with but w oul d n o t b e c AMP - induc i ble. EW S/A TF 1 approxima tel y 70 % inv olving t (X; 18 ) (p11 . 2; q11. 2) . do e s act ivate p romo ters w ith A TF 1 b indi ng s ites, Th is trans loca tion e v ent g e n er ates a fusion p rotein althoug h no t a ll such promo ter s wer e a ct ivated , from the 5 reg ion o f the ge ne a nd t he 3 9 SYT 9 and s ome p romo t ers w ere f ound t o b e r epres sed by 69± 71 region of or . Th er e i s no e vide n ce SSX1 SSX2 EW S/A TF 1 . Th erefore, EW S/A TF 1 m a y con t ribute of a trans cript b ei ng e xp res sed b y the r ecipri cal to m a ligna nt t rans formation b y several m e ch anisms. hybrid d e r ( 18 ) . Th e f unct ion o f the SYT gene is Fi rst, EW S/A TF 1 m a y co ns titut ivel y activate A TF 1 un k no wn , and s equen c e a nalysis revea ls no c l assical target g e n es that a re no rmally ind uc e d b y cA MP , or struc tur al mo tifs associated w ith D N A -bindi ng o r it m a y rep ress ge ne s that n or mally func tion i n trans cription a l regu l ation. How ever , the p resenc e o f gr owth c on trol. A lternat ivel y, EW S/A TF1 m a y acti- SH2 a nd S H3 d o ma ins sugg es ts that S YT mi ght vate n o v el g e ne s, perhaps gen e s regu l ated b y othe r func tion t hroug h protei n± protei n i nt eract ion. Th e CREB / A TF f amily me mb er s. rece nt i solation o f the m o u se h omo log o f SYT r e- In m o st MM S P t um o rs, two h ybrid transcr ipts vea led t ha t S YT i s ex pr es sed ubiquitous ly dur ing are gen e rated a nd e xp ressed b y the t (12 ; 22 ) ea rly emb ryogen es is, but e xp ression is res tricted (p13 ;q12 ) t ransloc ation. Th e e x pr es sion pro® le o f later in d e velopm e n t t o c a rtilage t issue, s peci® c n e u- the f usion gen e o n d e r (12 ) c h romos ome i s com p at- rona l ce lls a nd s ome e pithe lial-derived t issues . SYT ible w ith t he u b iquitous exp ression o f ATF . How- was also de tec table i n p rimary sperma toc ytes. ev er, this out -of-frame fu sion resul ts in a produ ct Sev eral studi es sugg es ted t hat S S con taine d t wo co ns isting o f the ® rst 65 N - ter minal amino a ci ds of di stinc t X chromo s ome b reakpoi nt s ites. How ever , ATF 1 , which is un l ikel y to b ind D N A or di me rize, the i den t i® c ation o f two c l os ely related g e ne s at ma king i ts role i n t rans formation u nc l ear. It i s un - Xp11 . 2 e stablished the i nv olveme n t o f di stinc t c od- likel y that e x pr es sion of the d e r(12 ) t rans cr ipt is ing r eg ions . De spite b ei ng 2 M b apart, and SSX1 es sential i n t rans formation g i ven r ep orts that 3 0 % of share 80 % homo logy . Bo t h e nc od e a 18 8 -aa SSX2 MM S P l ack ex p ression. protei n w ith a n N- ter mina l Krup pel-associated b ox (KRAB) t ha t i s thoug h t t o f unc tion a s a trans crip- 72,73 tion r ep ressor dom a in. Althoug h the se p rotei ns Desmo plas tic s mal l round cell t umor (DSR CT ) lack zinc ® ng e r m o tifs, the p resenc e o f the K RAB De s mop lastic small roun d c el l t umo r (DS RCT) i s sequen ce s sugg e st a role i n t rans cr iption. How ever , an agg r essive small roun d c e ll t um o r that o c cu r s this do ma in is not p resent i n t he c h imeric p rotein, pred om i na ntly i n a bdo mi na l ser osal surfaces and which s ugg es ts tha t S SX1 a nd S SX2 s eq uenc e s has a predi lec tion f or youn g m a les. Th e t um o r is a co nt ribut e t o t ransformation t hroug h no vel p rotei n± primitive s mall roun d c e ll w ith f eatur es of di verge nt protei n i nter action s or some o ther funct ion. SSX3, di fferen t iation, co -expr es sing e pithe lial, ne ur al and another KRAB p rotei n, is no t i mp lica ted i n t (X; my ogen i c m a rkers. Th e o rigi n o f this tumo r rema ins 18 ) -positive S S, but h as high h omo logy to S SX1 un c lear, b ut i t i s mo s t likely de rived f rom the and S SX2 ( 95 a nd 9 0 % , respectivel y). Th e s tud y of me s othelium . Almo st 1 0 0 % of these t umo r s co nt ain this ge ne m a y provide i ns igh t i nt o t he f unct ion o f a t(11 ; 22 ) (p13 ; q12) t rans loca tion t hat f uses the 5 9 SSX1 a nd S SX2. regi on o f the EWS gene to the 39 region of WT1, a tum o r suppres sor gen e i nv olved i n a subset o f 62± 66 Lipo sar comas ( LPS) Wilms ’ tum o rs. WT1 b inds DN A throug h a series of zinc ® ng e rs and r epresses the t rans cription Li posarco ma s (LS ) a re soft tissue t um o rs that o cc ur of ce rtain g e ne s. Th es e zinc ® nge r s are es sential f or primarily in t he e xt rem i ties a nd r et roperitone u m. transcr iption a l repres sion. Th e c hi me ric p rotei n Th ese t um o rs are from primitive m e s enc h ymal cells co nt ains the N - ter mina l reg ion o f EW S fused t o t he and t hey res emb le f etal adipose tissue. S ever al WT1 D N A -bindi ng d o m a in. Given t hat b oth the ch aracteristic c y tog e ne tic a berration s have been wild-type EWS ge ne a nd E W S fusion protei ns are ide n ti® e d f or adipose tumo r s. Th e m o st c o mm o n know n t o p artici pate i n t ranscr iption a l co mp lexe s, it LS are my xo id r oun d c el l l iposarcoma s, and g r ea ter is likely t hat E W S/W T1 fu nc tions as a trans cription than 90 % of my xo id l iposarcoma s co nt ain t he factor , possibly throug h WT1 t arget s. Th erefore , t(12 ; 16 ) (q13 ; p11) t rans loca tion i n w hich CHOP un l ike t he l os s of funct ion m u t ation i n W ilm’ s tu- on t he l ong a rm of chromo s ome 1 2 i s fused t o 22,75± 77 mo r , the l os s of the z inc ® nge r regi on o f WT1 i n FUS/TLS. Howe v er, this transloc ation e ven t EW S/W T1 s erves to c o nv ert W T1 f rom a repres sor has no t b een d et ect ed i n o t he r a dipose tum o r s and, of transcr iption t o a do mi nant t ranscr iption a l act iva- ther ef ore, ma y provide i nt eresti ng i ns ight i nt o t he tor on c og en e . trans formation p roce ss of thi s subset o f tum o rs. 82 T. J. Goletz et al. FU S/T L S is struc t ur ally similar to E W S ( . 50% and r ecog n ition h as incr ea sed c on side rably in t he amino a cid i de n tity) and i s exp ressed a t h igh lev el s last two d e ca des and h as bee n e xp er tly review ed 22 81 in a ll tissues exa mine d. TL S binds RNA and elsewhere. Br ie¯ y , T c el ls recog n i ze a ntige n s as en c od es a strong t rans cr iptiona l activation d om a in short pep tide s tha t a re boun d t o t he c e ll s urface i n in t he N - termi na l reg ion. Th er ef ore, like E W S, the c on t ex t o f ma jor histocom p atibility ( MH C) 81,82 1 FU S/T L S ma y funct ion a s a nu c lea r RNA -bindi ng molecules. In t he c a se of CD8 CTL , the T c el l protein. rece ptor (TC R) rec og n izes s hort p ep tide s (8± 10 CHOP , also ca lled G ADD 1 5 3 , is a me m b er of amino a cids) b oun d t o M H C class I mo lec ul es . the C CATT / e n h ance r-bindi ng p rotei n ( C/ E B P ) Th ese p eptide s are de rived f rom end o ge no u s ly ex - family of leu c ine z ipper t rans cr iption f actors that pressed p rotei ns which un de r go p roteol ytic p roce ss- regu l ate a dipocy te d i fferentiation. CHOP i s ex - ing i n t he c ytosol b y large p roteos ome c om p lex es . pres sed at l ow level s in a dipocytes; h owev er, mR NA Pe ptide f ragmen ts a re the n t rans ported i nt o t he levels i nc rease d u r ing c on di tion s of stres s such as lum e n o f the e nd o plasmic r eticul um (ER ) b y spe- DN A da mage . O v erexp ression o f CHOP i n N I H ci alized t rans porters o f antige n p roces sing ( TA P) . 3T 3 c el ls res ults in g r owth a rrest at G 1/ S . Thus, On c e i ns ide t he E R , pep tide s associate w ith a n CHOP i s thou gh t t o fu nc tion a s a do m i na nt n eg a - appropriate M H C cl ass I mo l ec ul e t ha t i s associated tive g r owth r egu l ator. with b eta-2- micr oglobul in ( b 2 m ) , an inv ariant s ub- In t he T L S /C HOP f usion p rotei n, the N - ter minal un i t w hich is thoug h t t o e n h ance e f ® ci ent M H C portion o f TL S is joined t o t he e nt ire C HOP c o di ng foldi ng , o p timi ze M H C/p ep tide b indi ng, and i n- 75,76 region. TL S /C HOP c an t rans form NI H 3T 3 cr ea se stability o f the M H C/p ep tide c o mp lex d ur ing ce lls a nd s tud i es i nd i ca te t hat t rans formation r e- trans port to a nd e xp ression o n t he c el l s urface. quires seq uenc e s from both T L S and C HOP . The Fo l low ing p eptide / M H C bind ing, the p eptide/ requirem e nt f or the C -termi na l leuc ine z ipper do - MHC/b 2 m co mp lexe s transverse t he E R and G olgi ma in of CHOP f or trans formation s ugg e sts a cruc ial apparatus, and a re di splayed o n t he c el l’ s surface role f or C/E B P p rotei n d ime rizati on. Althou gh it i s where t hey are subject t o s urveillance b y CTL . In un c lear w hether nor mal wild- type C HOP a ctivation the c ase o f CD4 Th cells, t he T C R reco gn i ze requires DN A -binding , the p oten t ial D N A -bindi ng sligh tly larger pep tide s (10 ± 25 a a) in t he c on tex t o f regi on , a basic reg ion o f the b ZIP d o m a in, is re- MH C class II mo lec ul es . Th ese p ep tide s are typi- quired f or trans formation. Th e r ole o f TL S se- ca lly de r ived f rom ma terial o r or ga nisms which have quen c es in t rans formation m a y be m o re t han that o f un d e rgon e e n d o/ p hagocy tos is by APC . Th us, in a strong t rans activator , since s ubstitut ion o f this ge ne r al, CD8 CTL rec ogn i ze i nt race llular ( en- regi on w ith o ther poten t t rans activating d o ma ins di d do ge n ou s ) peptides while C D4 T c el ls reco gn i ze no t m e di ate t rans formation . How ev er, substitut ions ex ter na l (ex o ge no u s) p rotei n f ragme nt s. with E W S seq uenc es wer e t ransforming . There- CTL ca n di sting u i sh self from no n- self peptide s fore, TL S /C HOP m a y con tribut e t o t rans formation associated w ith M H C cl ass I mo l ecu l es, so tha t by me ch anisms similar to t ho se p reviously di scu ssed ex pr es sion of viral protein s or alter ed c ellul ar in E W S fusion p rotei ns . protei ns will b e r e¯ e ct ed i n t he p eptide /M H C co m- plexe s displayed o n t he c e ll s urface. Althoug h the tum o r-speci ® c f usion p roteins de s cribed i n t hi s re- Poten t ial imm u n o t her apeut ic a pproach e s f or view func tion a s nu c lea r trans cr iption f actors, they the t reatmen t of p edi atric s arcom a s are still s ubject t o t he p roteo lytic p roces sing a nd Altho ug h mu l ti-mod ality therapy has imp roved s ur- present ation p athways de s cr ibed. Th er e i s exp er - vival rates for the p edi atric s arcoma s de scribe d i n ime n t al e vide n ce t ha t t um o r-associ ated n u c lea r thi s review , patien ts o ften r el apse, at w hich time protei ns , such a s mu t ant p 53, can i ndu c e i mm u n e 83± 88 responses to m u l ti-age nt c h emo t herapy are brief or responses. . no n -exi sten t . F u r ther mor e, patien ts w ho present Th e i de nt i® c a tion o f TA A and a n incr ea sed with m e t astatic d i sease at d iagn o sis do v er y poorly un d e rstand i ng o f the r eq uireme n ts f or the i nd u c tion in s pite o f agg r es sive mu l ti-moda lity ther apy. Th ere- of ce ll-medi ated i mm u n e r esponses (Ta ble 2) h as fore, efforts are ne e de d t o d e velop n o vel t reatme nt s, led t o a dvances in i mm u n o ther apy. While a num- such a s imm u no t he rapies . Stud i es o v er the p ast ber of TA A have been i de nt i® e d f or sev er al tumo r 90± 93 de c ade h ave provided e vide n ce t hat t rea tme n ts types, it i s un cl ea r whet he r a ll TA A will b e based on t he m a nipulation o f the i mm u n e s ystem ef fective t umo r regr ession a ntige ns . Ide ally, one ca n me di ate r egr ession o f es tablished m e tastat ic wou l d l ike t o i den t ify and t arge t T A A which play a ca ncer. M o re s peci® c a lly, cel l-med iated i mm u n i ty key role i n n e o plastic t rans formation, so that t hey ca n play a cr itical r ole i n t um o r regr ession. ca nno t b e l ost w ithou t l oss of ma lign a ncy . Th e T l ymphocy tes are mo s t often c atego r ized a s tum o r-associ ated t rans loca tions ide nt i® e d f or a 1 1 CD8 cy tot oxi c l ymphocy tes (CT L ) o r CD4 nu m b er of ped iatric s arcoma s such as ES and A R helpe r l ymphocytes (Th ), and b oth t ypes of T c el ls ma y ver y wel l b e s uch a ntige ns , since t hey ge ne r ate are kno wn t o p lay a role i n t um o r reg res sion. Ou r func tion al ch imer ic t rans cr iption f actor s kno wn t o un d e rstand ing o f antige n p roce ssing, pres en tation , co nt ribut e t o a bberrant g en e e xp ression. Mo re Mo lecular alterati ons in pedi at ric sarcomas 83 Table 2. Imm u notherap eutic ap proach es using tumor-as sociated a n tigens Ac tive i mm u n otherap y u s ing i mm u nod omi nant p eptides : alone with a dju v ant s linked t o h elper peptide s Ad m i nistered : in l ipids/l iposom e s pulsed o nt o a ntigen- present ing cells ( APCs) Substit ut ed p eptide s imm u no dom i na nt p eptides w ith a mi no a cid s ubstitu tions t o i ncrease bind ing to M H C Proteins alone with a dju v ant s DNA `na ke d’ DNA e nc odi ng c ancer a nt igens a dmi nistere d u sing gene g un int ramu s cul ar i njection associa ted/ l inked t o l ipids Re comb ina nt v iruses recom b inant v irus es, s uch a s v accinia , f owlpox or a de nov irus, e ncod ing cancer a ntigens , a lone or i n c om b ination w ith g enes e nc od ing cytok ines c os timu l atory m o lecul es o r imm u nos timu l atory fa ctors Re comb ina nt b acteria recom b inant b acteria s uch a s b acillus c alme tte± g uerin ( BC G), Salmonella or Listeria enginee red t o e xpress c anc er antigens alone o r w ith g enes e nc odi ng c ytokin es, c ostimu latory mo lecul es o r other i mm u no stimu latory f actor s Ac tive i mm u n otherap y f ollow ed b y c ytokine s Int erleuk in 2 (IL-2), IL-6, IL-10, IL-15 Passive i mm u not herapy w ith a nt i-tumo r l ymp hocyte s g enerate d in vitro Ge neratio n o f CTL us ing i mm u no domi nant p eptide -pulsed A P Cs Ge neratio n o f Th b y c oincub ation o f APC with a ntigeni c p eptides speci® c a lly, the b rea kpoint ju nc t ions are likely n eo - do es not r eact a ga inst all possible antige ni c d e ter mi- antige ns . Fu r ther , it s hould b e p ossible to a void na nts, ch aracterizati on o f the i mm u no d o mi na nt autoimm u n e r esponses by focus ing o n m i ni ma l pep- pep tide s in t he t um o r reg res sion antige ns will f ur- tide s cor respondi ng t o t he s eq uenc es which span the ther aid in t he d e vel opme n t o f effective t reatme nt s. brea kpoint , s ince t hes e wou ld n o t b e p resen t i n Th e i de nt i® c ation o f TA A and t he c l on ing o f the no r mal ce lls. Th is hypothes is was tested i n a nimal ge ne s which enc o de t hem p rovides num e r ous op - mo d e ls u sing s ynthe tic p eptides co rres pond i ng t o portun i ties for the d ev elop ment o f ca ncer t her apies the b reakpoi nt j unc tion s in E S and A RMS as im- (Ta ble 2) . T h erapies co ul d u t ilize t he T A A protein mu n o ge n s. In t hes e studi es , pep tide -pul sed A PC ei ther a lone o r with a djuvants. Alterna tivel y, the admi ni ster ed i nt raveno us ly, ge ne rated C D8 CTL admi ni stration o f peptides de rived f rom the T A A responses ca pable of lysing p ep tide -pulsed t um o r protei n a dminister ed a lone, with a djuvants or in cells in vitro as well a s tumo r ce lls t rans fected t o co mb inatio n w ith h elpe r p ep tide s , has cer tain a d- ex p ress the f ull-leng t h f usion p rotein. Fu rther more, vantage s in t hat t his approach has bee n d e mo n- the se r esponses were a ble to r ed uc e o r irradicate strated t o g e n er ate T c e ll r es pons es while h aving tumor . Th es e data de m o n s trate t hat t he mi ni ma l risk in t he i nd u ct ion o f un wa nted a nd in vivo ch imer ic f usion p roduc t s res ulting f rom ch romo- potent ially da nge rous autoimm u n e r eact ions . Anti- somal trans loca tions can s erve as neo antige ns . tum o r responses ge ne r ated b y peptide v accina tion Be ca use the t rans loca tion e ven t s are tumo r speci® c , ma y be a ugme n t ed b y ma nipulation o f the r out e/ the rapies targe ting t he r es ulting f usion p roteins mo d e o f admi ni stration. Th e c lon i ng o f ge ne s en- woul d b e h igh ly speci ® c a nd p otent ially les s toxi c. co di ng T A A will fa cilitate t hei r exp ression i n Clini ca l trials are cu r rent ly u nd e rwa y in p atien ts high- ef® ci enc y e xp ression s ystems, such as rec om b i- with E S and A RMS to e valua te t he g en e ration o f na nt v iruses or bacter ia. Th ese v ec tor s ca n be e ng i - anti-tum o r responses using a similar approach . In ne er ed t o e x pr es s the T A A alone o r in c onj unc tion addi tion , stud i es a re on g oi ng t o n o t o nl y ide nt ify with c ytokin e g en e s or ge ne s enc od i ng c os timul a- addi tion al TA A, but a lso to g ain a n un de r standi ng tor y mo l ec ul es . Fu r ther mor e, direc t i njec tion i nt o as to w hich T A A ma y serve as tum o r reje ction mu s cl e o f DN A enc od i ng a ntige ns or the u se o f antige ns . Sinc e i t i s cl ear tha t t he i mm u n e s ystem `ge n e g un s ’ in w hich DN A is attache d t o s mall 84 T. J. Goletz et al. particl es t hat a re me ch anically propelled i nt o c el ls promo t er r egi on f rom one o f the p artner ge n es . is also an ef fective m e thod o f ind u ci ng i mm u ne Howe v er, this me c hani sm has no t b een o bserved i n 95± 100 responses. solid tumo r s, but m a y be r el evant i n h ema topoiet ic Ant i-tumo r res pons es have been g e ne r ated b y in ma lign a ncies . No ne t he les s, it i s likely that e x - vitro sens itizati on o f peripheral blood l ymphocytes pression of hybrid proteins in s olid t um o r s dysregu- (PB L ) t o p ep tide -pul sed A PC or irradiated t um o r lates the t rans cr iption o f key gr owth c o nt rol g e ne s or ce lls. Repea ted i n v itro sens itizati on u s ing i m- pathways, ther eb y promo t ing t umo r igen e sis. mu n o do m i na nt p eptides from me l anoma antige ns While f usion p rotein s are likely t o i nvoke a com b i- pulsed o n t o a utolog o us peripheral blood m o n on u - na tion o f the a forem e nt ione d m e c ha nisms, the r e- cl ea r ce lls i n t he p res en ce o f IL-2 resul ted i n t he du n d anc y o f thei r role i n o nc og e ne sis is no t ewo r thy. ex p ansion o f CTL (10 , 00 0 - fold) o ver a 6- week pe- Th e m u l tiple i nt er ch ange o f func tion a l do ma ins riod. Cel ls ge ne rated b y this approach showed i m- from related g e ne s such a s FLI1 and ERG in mu n e r ea ctivity 50 ± 10 0 t ime s gr ea ter than PNETs, PAX3 and PAX7 in ARMS and SSX1 and co rres pond i ng t um o r in® l trating l ymphocytes SSX2 in S S res ult i n s imilar tum o r pheno t ypes. (TIL) and s peci ® c ally rec og n ized t he a ppropriate Do m a in-swap exp erime n ts i nv olving E W S for TL S imm u n o do m i na nt p eptide a s well a s tum o r ce lls a s in T L S /C HOP s howed t hat s ubstitut ions ca n be me a sured b y lysis and c y tokine r el ea se. Stud ies i n ma de w ith l ittle c hang e i n m o rpholog y. H ow ever , ex p erime n tal a nima l mo d e ls s ugg e st t ha t s peci® c ot her e x p erime n ts i n w hich FL I 1 w as ex ch ange d f or tum o r reco gn i tion a s det ermi ne d b y lysis and c y - CHOP i n f usions with T L S or EW S had an ef fect tok ine s ec retion a ssays cor related h ighl y with in vivo on c e ll m o rphology , such that t he m o rpholog y in anti-tum o r effect s. Th es e co rrel ates h ave also some c a ses was de p end e nt o n t he D N A -bind i ng been o bserved i n p atient s treated w ith a utologo us regi on o f the c hime r ic t rans cription fa ctor. F i na lly, 103,104 TIL. In s everal o t her stud ies, T c el ls stimu- of no te i s the e a rly ons et o f ma ny of the se t um o rs. lated in vitro wer e c apable o f recog n i zing a nd l ysing Th is sugg e sts that t he g e ne s inv olved i n s arcoma -as- target c e lls p ulsed w ith p ep tide s know n t o b ind t o a soci ated t ransloc ation s have speci® c p atter ns of de - particu lar MH C cl ass I mo lecu le; h owe v er, thes e vel opme n t al r eg u lation , and t hat d ysregu lation o f same T c el ls were o f ten i nc apable o f recog n izing this temp oral regu l ation h as profoun d e f fects. and l ysing t he l ow levels o f proces sed p ep tide s ex - Attem p ts at d ev elop ing n e w t her apeut ic a p- pres sed by tumo r cel ls. Th us, ther e i s co ns ider - proach es to t he t rea tme n t o f thes e tum o rs have able h eter ogen e ity i n a nti-tum o r responses . inc l ud ed i mm u n o ther apy. Howe ver , succe ssful i m- mu n o ther apeut ic s tratagi es mu st m e e t s ever al cri- teria, t he ® rst of which is the e xp ression o f TA A tha t Summary are rec og ni ze d b y T l ymphocy tes . In t he c a se of the Th e g e ne ration o f chi me ric t rans cr iption f actors is a sarcoma s present ed i n t his review , the c hi me ric co mm o n c on sequen c e o f ch romos oma l trans loca - trans cription fa ctors represent p oten t ial T A A. Stud- tion s in s olid t um o r s. Th e r esulti ng f usion p roteins ies in e x p erime n tal a nimals sugg es t tha t t he t rans lo- have been s hown, in s ev eral ca ses, to h ave trans - ca tion b rea kpoints in E S and A RMS rep resen t forming a ctivity . Chime ric o n co proteins ma y func - ne oa ntigen s which can b e r ec og ni zed b y CTL . Fu r - tion t hroug h sev er al me ch anisms. Fi rst, a strong ther mor e, these r esponse w er e s uf® cient t o m e di ate act ivation d o ma in from one g e ne m a y be f used to in vivo tum o r reg ression i n a nima l mo de l s. Clini cal the D N A -bindi ng s peci ® c ity r egi on o f another ge ne , vacci ne s tud i es are on g oi ng t o e v aluate t he a bility of leadi ng t o d y sregu l ated e xp ression o f targe t g en e s. thes e TA A to s erve as tum o r regr ession a ntige ns . Th e f usion p rotei ns associated w ith M M S P, ARMS , Fi nally, iden t i® c ation o f the i mm u n o do mi na nt e p i- and P N E T s are exa mples of this me cha nism. How- top es in t um o r reg r es sion antige ns will f avor the ev er, in m y xo id l iposarcoma , the F U S/C HOP g e ne ind u c tion o f ef fective a nti-tu mo r responses. Scr een- produ ct a ppears to m e di ate i ts effect o n t ranscr ip- ing v accine s and v arious de livery systems (pept ide s tion t hroug h protein ± p rotei n i nter actions and m a y or proteins in a djuvants or on d en d ritic c e lls, DN A , no t r eq uire D N A -binding. Sec ond , a fusion partne r viruses ) in a nima ls, such as HLA -transgen ics, will ma y con tribut e m o re t han an act ivation d o m a in. hel p to i den t ify the m o st p romising v accine s for us e Fo r exa mple, the E W S/F L I 1 f usion protein o f ES in c l ini ca l trials. seem s to c o mb ine t he t ransactivati on d om a in of EW S with t he D N A -binding r egi on o f FL I 1. How- References ev er, the f usion p rotein a ppears to m e di ate n o vel protein± protein /p rotei n± nu c leic a cid i nt er actions . 1 Ra bbitts T H. Chromo soma l t ransloc ation s i n h um a n Also, the c hi me ric o n c op rotein m a y het er odi me rize cancer. 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