Braf V600E cooperates with Pten loss to induce metastatic melanoma

Transcription

1 Brf V6E oopertes with Pten loss to indue metstti melnom Dvid Dnkort 1,5,6, Dvid P Curley 2,6, Roert A Crtlidge 1, Betsy Nelson 2, Anthony N Krnezis 3, Willim E Dmsky, Jr 2, Mingjin J You 4,5, Ronld A DePinho 4, Mrtin MMhon 1 & Mrus Bosenerg 2,5 Muttionl tivtion of BRAF is the erliest nd most ommon geneti ltertion in humn melnom. To uild model of humn melnom, we generted mie with onditionl melnoyte-speifi expression of BRf V6E. Upon indution of BRf V6E expression, mie developed enign melnoyti hyperplsis tht filed to progress to melnom over 15 2 months. By ontrst, expression of BRf V6E omined with Pten tumor suppressor gene silening eliited development of melnom with 1% penetrne, short lteny nd with metstses oserved in lymph nodes nd lungs. Melnom ws prevented y inhiitors of mtor1 (rpmyin) or MEK1/2 (PD32591) ut, upon esstion of drug dministrtion, mie developed melnom, inditing the presene of long-lived melnom-inititing ells in this system. Notly, omined tretment with rpmyin nd PD32591 led to shrinkge of estlished melnoms. These mie, engineered with ommon geneti profile to humn melnom, provide system to study melnom s rdinl feture of metstsis nd for prelinil evlution of gents designed to prevent or tret metstti disese. Mlignnt melnom is noted for its ggressive linil ehvior, propensity for lethl metstsis nd therpeuti resistne. This linil piture, oupled with n inrese in inidene, hs motivted efforts to understnd the geneti underpinnings of melnom initition nd progression nd trnslte suh insights into effetive preventive nd therpeuti strtegies 1,2. Severl key geneti lesions governing melnom initition nd progression hve een identified, the erliest nd most ommon eing point muttion (T1799A) in the BRAF proto-onogene, whih is deteted in ~65% of ffeted individuls 1,3 7. BRAF T1799A enodes BRAF V6E, onstitutively tive protein serine kinse tht eliits sustined tivtion of the BRAF MEK1/2 ERK1/2 MAP kinse pthwy. Although it ontriutes to the errnt pthophysiologil hrteristis of the melnom ell 8,9, onogeni tivtion of BRAF in melnoytes is insuffiient for full mlignnt onversion owing to tivtion of senesene 1,11. Hene, progression to mlignnt melnom is invrily ompnied y silening of one or more tumor suppressor genes, most ommonly PTEN or CDKN2A 5, The omintion of mutted BRAF nd silening of PTEN expression is ommon in humn melnom (~2%), lthough geneti evidene for BRAF nd PTEN oopertion in this disese hs not een otined to dte To understnd nd vlidte the role of suh geneti events nd their intertions in melnom initition, progression nd response to therpy, we hve generted onditionl mouse model of Brf V6E - indued, Pten-defiient metstti melnom. This geneti model repitultes key pthophysiologil spets of the humn disese nd offers ontrol over the timing nd lotion of melnom initition. Moreover, we demonstrte the utility of this model in the evlution of phrmologi gents tht trget key signling pthwys linked to these signture events in the melnom ell, supporting the utility of this disese model in prelinil experimentl therpeutis. RESULTS BRf V6E promotes enign melnoyti hyperplsi in vivo Brf CA mie hror germline onditionl Brf V6E llele, the expression of whih is initited t physiologil levels under the ontrol of the gene s hromosoml promoter y the tion of Cre reominse (Fig. 1) 17. When the Brf CA llele ws omined with onstitutively tive Cre trnsgene under the ontrol of the Tyr promoter (Tyr::Cre, Te1) no vile offspring were otined (Supplementry Tle 1 online) 18. Similrly, ompound Brf CA nd Dt promoter direted Cre (Dt::Cre) trnsgeni mie suumed within 3 months of ge 19 (D. Mtzen et l., unpulished dt). To irumvent lethlity ssoited with emryoni expression of Brf V6E, we used Tyr::CreER trnsgene, whih enodes onditionlly tive CreER T2 speifilly in melnoytes (Fig. 1) 2. In the sene of 4-hydroxytmoxifen 1 Cner Reserh Institute & Deprtment of Cell nd Moleulr Phrmology, Helen Diller Fmily Comprehensive Cner Center, University of Cliforni, Sn Frniso, Cliforni, USA. 2 Deprtment of Pthology, University of Vermont College of Mediine, Burlington, Vermont, USA. 3 Cner Reserh Institute & Deprtment of Pthology, Helen Diller Fmily Comprehensive Cner Center, University of Cliforni, Sn Frniso, Cliforni, USA. 4 Belfer Institute for Applied Cner Siene, Deprtments of Medil Onology, Mediine & Genetis, Dn-Frer Cner Institute nd Hrvrd Medil Shool, Boston, Msshusetts, USA. 5 Present ddresses: Deprtment of Biology, MGill University, Montrel, Cnd, H3A 1B1 (D.D.); Deprtment of Hemtopthlogy, Division of Pthology nd Lortory Mediine, University of Texs, M.D. Anderson Cner Center, Houston, Texs, USA (M.J.Y.); Deprtment of Dermtology, Yle University Shool of Mediine, New Hven, Connetiut, USA (M.B.). 6 These uthors ontriuted eqully to this work. Correspondene should e ddressed to M.M. (mmhon@.usf.edu) or M.B. (mrus. osenerg@yle.edu). Reeived 12 August; epted 13 Ferury; pulished online 12 Mrh 29; doi:1.138/ng volume 41 numer 5 my 29 nture genetis

2 d e Allele Tyr::CreER Brf CA/+ Pten flox/flox CreER BRf WT Pten WT Expressed proteins 4HT Ative CreER BRf V6E No Pten produed i ii iii iv Figure 1 Benign hyperplsis indued y melnoyte-speifi expression of BRf V6E. () Mie rrying vrious onditionl lleles of Brf (Brf CA ) nd/or Pten (Pten lox4-5 or Pten lox5 ) were rossed to Tyr::CreER mie with melnoyte-speifi expression of hormone-dependent form of Cre reominse (CreER T2 ) 17,2. Ativtion of CreER y 4-HT leds to melnoyte-speifi onversion of Brf CA to Brf V6E nd the onversion of the Pten lox lleles to null lleles. ( d) Brf CA/+ (), Tyr::CreER; Brf CA/+ () nd Tyr::CreER; Brf CA/CA (d) mie were treted topilly with 4-HT nd monitored for 75 weeks for signs of melnoyti prolifertion (i). Mie were euthnized nd skin from the er (ii, iii) nd flnk (iv) ws stined with hemtoxylin nd eosin nd inspeted for the presene of pigmented ells. (e) A Tyr::CreER; Brf CA/+ mouse developed ppulr pigmented lesion ~6 weeks fter topil dministrtion of 4-HT (i). This mouse ws euthnized nd skin setions enompssing the lesion were stined with hemtoxylin nd eosin nd inspeted for the presene of pigmented ells (ii, iii). Sle rs: ii, 2 mm; iii,.1 mm. (4-HT), Tyr::CreER; Brf CA/+ mie did not show ny disernle phenotype over 18 months of oservtion (n 5, Fig. 1, i). However, topil dministrtion of 4-HT to either fur ering or glrous skin or systemi 4-HT dministrtion led to the pperne of highly pigmented lesions tht were deteted y dys fter 4-HT dministrtion (Fig. 1, i). Suh lesions were not deteted in 4-HT treted Tyr::CreER mie, inditing tht they require the onversion of the Brf CA llele to Brf V6E (Supplementry Fig. 1 online) 2. Brf V6E -indued pigmented lesions were frequently loted t the derml-epiderml juntion with extension into the dermis, s is seen in humn ompound melnoyti nevi (Fig. 1, iii). Other melnoyti prolifertions formed in the dermis with no pprent juntionl omponent. Brf V6E -indued pigmented lesions ppered only in regions of the skin to whih 4-HT ws pplied nd were not indued y topil dministrtion of solvent s ontrol (Supplementry Fig. 1). Osionlly, when high onentrtions of 4-HT were topilly dministered to mie, we oserved perioulr lesions on the fe nd on the glrous skin of the pws, most likely owing to grooming ehvior (Supplementry Fig. 1,). However, y reduing the mount of 4-HT dministered, we ould eliit sptilly restrited melnoyti lesions with no evidene of inpproprite spreding to untreted sites. To determine whether BRf V6E -indued pigmented lesions would progress spontneously to mlignnt melnom, we monitored 35 4-HT treted Tyr::CreER; Brf CA mie over 15 2 months. Although two mie developed smll ppulr pigmented lesions, these lesions were not onsidered mlignnt in nture given their lk of signifint invsion or metstsis, the smll nd self-limited tumor growth, the nl ytologi fetures of the ells nd the sene of errnt mitoti figures within the lesions (Fig. 1). These dt re onsistent with the hypothesis tht BRAF V6E expression in humn melnoytes promotes n initil phse of prolifertion tht is susequently restrined from mlignnt progression y enggement of ell yle rrest progrm with fetures of senesene, refleting the linil oservtion of growth rrest of pigmented nevi in humns 1,21. In ddition, we generted smll numer of Tyr::CreER mie tht were homozygous for Brf CA to ssess the impt of two opies of the Brf V6E llele (n = 6). Topil 4-HT dministrtion to the er, til or flnk of these mie led to the pperne of enign melnoyti hyperplsis tht were onsiderly lrger nd more highly pigmented thn in mie heterozygous for Brf CA expressing only one opy of Brf V6E (Fig. 1d, i iv, respetively). Indeed, this phenotypi presenttion ws so reproduile tht mie ould e genotyped (Brf CA/+ versus Brf CA/CA ) on the sis of the size nd pigmenttion of the melnoyti lesions tht developed. These dt indite tht Brf V6E gene dosge exerts physiologilly relevnt impt on melnoyte prolifertion, n experimentl oservtion tht my rtionlize the oservtion of inresed opy numer of onogeni BRAF in humn melnom nd other tumors 22,23. BRf V6E oopertes with Pten loss to indue metstti melnom Humn melnoms expressing BRAF V6E often show tivtion of the PI3 -kinse PDK AKT pthwy through silened expression of the PTEN tumor suppressor gene 14,15,22. To model this geneti profile, we generted Tyr::CreER; Brf CA mie tht were lso homozygous for either one of two different onditionl Pten lleles. One llele (Pten loxp-neo ) onditionlly deletes Pten exons 4 nd 5 (ref. 24). The seond llele (not previously desried) permits Cre-medited deletion of Pten exon 5. We refer to these lleles s Pten lox4-5 nd Pten lox5, respetively. Notly, the tion of Cre reominse on oth lleles leds to deletion of exons tht enode mino ids essentil for the Pten s PI3 -lipid phosphtse tivity 24. In ontrst to Brf V6E -indued melnoyti neoplsi (Fig. 1), 4-HT tretment of Tyr::CreER; Pten lox4-5/lox4-5 or Tyr::CreER; Pten lox5/ lox5 mie filed to eliit ny melnoyti phenotype over ~18 months (n = 2). These dt re onsistent with oservtions tht Dt::Cre; Pten lox/lox mie do not develop melnom unless treted with rinogen 25. However, 7 1 d following 4-HT dministrtion, ll Tyr::CreER; Brf CA ; Pten lox/lox mie showed mrked expnsion of highly pigmented ells regrdless of where the 4-HT ws dministered to the skin nd regrdless of whih Pten llele ws used (Fig. 2 ). Indeed, the oopertion etween Brf V6E nd Pten silening ws so strong tht onfluent melnoyti prolifertion ws oserved in 4-HT treted regions of the skin of Tyr::CreER; Brf CA ; Pten lox/lox mie. By ontrst, tretment with dimethylsulphoxide solvent ontrol ws without effet (Fig. 2 nd Supplementry Figs. 2 nd 3 online). Arogtion of Pten expression ws onfirmed y protein lotting nlysis of extrts of primry lesions derived from nture genetis volume 41 numer 5 my

3 d e f g h i m j k l Survivl (%) Brf CA Pten lox/lox Brf CA ; Pten lox/lox HT treted Tyr::CreER; Brf CA ; Pten lox5/lox5 mie (Supplementry Fig. 4 online). Pigmented melnom ells were found throughout the dermis nd suutis nd showed pgetoid spred into the epidermis (Figs. 2h,i,k). Moreover, there ws rpid emergene of highly pigmented mlignnt lesions eliited y 4-HT in the fe, flnk or til skin, the lotion of whih ould e highly restrited y topil dministrtion of low-dose 4-HT (Fig. 2d f nd Supplementry Fig. 3,). These lesions progressed rpidly to dvned mlignny suh tht ll Tyr::CreER; Brf CA ; Pten lox/lox mie required euthnsi 25 5 d following 4-HT dministrtion (Fig. 2m). Histologil nlysis of primry skin lesions reveled highly pigmented ells with vrily shped enlrged nulei ontining lumped hromtin, prominent nuleoli nd typil mitoses (Fig. 2l). In ddition, the melnoyti origin of ells in the lesions ws onfirmed y the presene of undnt melnin nd y immunostining with ntiody to PEP1 to detet expression of tyrosinse-relted protein 1 (Fig. 2j,k) 26. To further doument the rte-limiting role of Pten in onstrining mlignnt progression, we followed smll numer of 4-HT treted Figure 2 BRf V6E oopertes with Pten loss in the indution of mlignnt melnom. ( ) Tyr::CreER; Brf CA/+ ; Pten lox5/lox5 mie were treted topilly on the pw with 4-HT to eliit Brf V6E nd to silene Pten expression. The presene of pigmented lesions ws ssessed 6 (), 8 () nd 1 () dys following 4-HT dministrtion. (d l) Tyr::CreER; Brf CA/+ ; Pten lox4-5/lox4-5 mie were treted topilly with 4-HT on the right er nd flnk. Mie were monitored for 7 weeks. Mie were euthnized t ~7 weeks with the presene of mlignnt melnom lesions ssessed y visul inspetion of the underside of the ventrl/ lterl skin. Tyr::CreER; Brf CA/+ ; Pten lox5/lox5 mie were treted topilly on the er, flnk nd til with 4-HT. Twenty-five dys lter, mie were euthnized nd skin setions were prepred, stined with hemtoxylin nd eosin nd exmined for the presene of pigmented ells. Almost onfluent prolifertion of densely pigmented ells ws oserved in the er nd skin (g) tht penetrted deep into the dermis nd the suutis (h,i) nd showed signs of pgetoid spred into the epidermis (h, k). Cells in these lesions stined positive with ntiser to PEP1 tht detets expression of Tyrp1 (j,k). (l) Pigmented ells in the lesions displyed histologil hrteristis of mlignnt melnom, inluding mrked ytologil typi, prominent nuleoli nd errnt mitoti figures. (m) Kpln- Meier survivl nlysis of 4-HT treted Tyr::CreER; Brf CA/+ ; Pten +/+ (n = 22), Tyr::CreER; Brf +/+ ; Pten lox4-5/lox4-5 (n = 5) nd Tyr::CreER; Brf CA/+ ; Pten lox4-5/lox4-5 (n = 22) mie. Log rnk tests of survivl plots of the dt indited sttistilly signifint differene etween the following survivl urves: Tyr::CreER; Brf CA/+ ; Pten +/+ versus Tyr::CreER; Brf CA/+ ; Pten lox4-5/lox4-5 (P <.1) nd; Tyr::CreER; Brf +/+ ; Pten lox4-5/lox4-5 versus Tyr::CreER; Brf CA/+ ; Pten lox4-5/lox4-5 (P <.2). Tyr::CreER; Brf CA mie tht were heterozygous for the Pten lox4/5 llele. These mie initilly developed enign melnoyti lesions similr to those oserved in Tyr::CreER; Brf CA mie. However, they eventully developed fol mlignnt melnoms tht we elieve re most likely due to stohsti silening of the wild-type Pten llele, s hs een oserved with other tumor suppressor genes (Supplementry Fig. 2). Brf V6E ; Pten / melnom ells re invsive nd metstti At euthnsi Tyr::CreER; Brf CA ; Pten lox/lox mie were sujeted to omplete histopthologi survey for evidene of melnom invsion nd metstsis. As desried ove, ll mie nlyzed hd evidene of melnom ells throughout the dermis with evidene of invsion into the suutis (Fig. 2). In ddition, spred of melnom ws oserved into the regionl drining lymph nodes in 1% of mie, with expnsile growth noted in most mie (Fig. 3 ). Metstses were lso noted in mie with lolized tumors in the distl region of the til tht hd ler evidene of pigmented ells in the ili lymph nodes (Fig. 3d nd Supplementry Fig. 3) nd lungs (dt not shown). Exmintion of d e f g Figure 3 BRf V6E oopertes with Pten loss in the indution of invsive nd metstti melnom. () Tyr::CreER; Brf CA/+ ; Pten lox4-5/lox4-5 mie were treted topilly on the er, flnk nd til with 4-HT; six weeks lter mie were euthnized nd inspeted for evidene of pigmented ells in the mmmry glnd lymph node. (,) Tyr::CreER; Brf CA/+ ; Pten lox5/ lox5 mie were treted similrly to those desried in, euthnized nd then inspeted y mirosopy t low () nd high () power for the presene of pigmented ells in the lymph nodes. (d) A Tyr::CreER; Brf CA/+ ; Pten lox5/lox5 mouse ws treted topilly with 5mM 4-HT in 1% (v/v) ethnol on the distl til. The mouse hd visile tumor within 4 weeks ut did not require euthnsi until 24 weeks lter fter 4-HT dministrtion. The presene of pigmented ells in the ili, lumr, inguinl, xillry nd sumndiulr lymph nodes ws ssessed y stereomirosopy. Tumor ells were restrited to the ili nodes (pitured). u, uterus; i, right ommon ili rtery. (e) Tyr::CreER; Brf CA/+ ; Pten lox4-5/lox4-5 mie were treted topilly on the er, flnk nd til with 4-HT. Mie were euthnized 6 7 weeks fter 4-HT tretment t whih times the lungs were exised, lered of lood nd visully inspeted for the presene of pigmented lesions. (f) The insert in e is shown t higher mgnifition in f. (g) Lung setions were prepred, stined with hemtoxylin nd eosin nd exmined for the presene of pigmented ells in the lung prenhym. 546 volume 41 numer 5 my 29 nture genetis

4 Survivl (%) Vehile Time fter Cre tivtion t = (wk) Vehile 2 Rp/Veh tretment (21 d) Time fter Cre tivtion (d) Figure 4 Prevention of BRf V6E -indued melnoms y rpmyin. () Adult Tyr::CreER; Brf CA/+ ; Pten lox5/lox5 mie were treted topilly on the er, flnk nd til with 4-HT. The next dy, mie were rndomly ssigned to e dministered rpmyin (7.5 mg/kg, n = 5) or solvent ontrol (n = 4) for 21 d. Mie were monitored dily for the presene of utneous mlignnt melnom nd euthnized ording to stndrd ody onditioning sore. After three weeks of rpmyin tretment, drug dministrtion ws esed nd mie were monitored for the presene of utneous mlignnt melnom s desried ove. Mouse survivl ws plotted using Kpln-Meier survivl urve. () Representtive imges of vehile ontrol (left) or rpmyin (right) treted mouse immeditely fter the end of the 21-d drug dministrtion re presented. () Kpln- Meier survivl urve of vehile (n = 4) or rpmyin (n = 5) treted ohorts of mie nlyzed in this experiment. All mie were treted for the indited 21 d s indited in. the lungs reveled visile evidene of t lest one nd, in mny ses, multiple nests of metstti melnom ells (Figs. 3e g). These dt indite tht tivted Brf V6E nd Pten defiieny ooperte to produe primry utneous mlignnt melnom with roust pity to invde into the skin nd to metstsize to drining lymph nodes nd distl orgns tht re ommonly trgeted in the humn disese. Prevention nd therpy of BRf V6E -indued melnoms y phrmologil inhiition of MEK1/2 nd mtor1 Melnom is noted for its resistne to hemotherpy, lthough there re nedotl reports of drmti responses to therpy trgeted ginst mutted KIT (-KIT) in humns 13,27. Consequently, we wished to determine whether trgeted inhiition of downstrem effetors of BRf V6E or PI3 -kinse signling ould prevent the development of melnom in this model system. To do so, we used speifi nd seletive inhiitors of MEK1/2 (PD32591) or mtor1 (rpmyin), whih re downstrem of BRf V6E or PI3 -kinse, respetively 28,29. Melnom ws initited in dult Tyr::CreER; BRf CA ; Pten lox/lox mie y topil dministrtion with 4-HT. Either 1 d (rpmyin, Fig. 4) or 1 week (PD32591, Fig. 5) lter the mie were treted systemilly either with the relevnt solvent ontrol or with rpmyin (7.5 mg/kg) or PD32591 (15 mg/kg) for 3 or 6 weeks, respetively. Solvent-treted mie rpidly developed melnom tht required euthnsi within 3 6 weeks (Figs. 4 nd 5, pnel i). By ontrst, tretment with either rpmyin or PD32591 mrkedly inhiited melnom formtion, suh tht y the time ll of the solvent-treted mie were euthnized, drug-treted mie were live nd showed no signs of melnom formtion (Figs. 4 nd 5, pnel ii). To determine whether tretment with either rpmyin or PD32591 hd ompletely eliminted melnom ells initited y 4-HT dministrtion, we esed drug tretment in oth groups of nimls nd monitored the nimls for n dditionl 3 11 weeks (Figs. 4 nd 5). Regrdless of the drug used, ll of the nimls susequently developed mlignnt melnom requiring their euthnsi within 5 11 weeks following esstion of drug dministrtion (Figs. 4 nd 5 (ottom), ). These dt indite tht the prevention of melnom in the drugtreted mie ws not result of tehnil flw in the tivtion nd funtion of CreER following 4-HT dministrtion. Moreover, these dt re onsistent with the presene of ltent melnom-inititing ells tht rise shortly fter the CreER tivtion nd tht n survive extended exposure to gents tht trget signling pthwys essentil for the melnom ell division yle nd survivl. To determine whether melnoms tht reur following esstion of MEK1/2 inhiitor tretment re resistnt to retretment of mie with this gent, melnom ws initited in ohort of 1 3-weekold Tyr::Cre; Brf CA ; Pten lox4-5/lox4-5 mie. Ten dys lter, ll mie were treted systemilly with PD32591 for period of 6 weeks. At this time, onsistent with results desried ove, none of the mie showed ny signs of melnom. Upon esstion of PD32591 dministrtion, the mie were left untreted for period of 8 weeks. At this time mie were rndomized into two groups of five, one of whih ws dministered solvent ontrol (group A) nd the other PD32591 (group B) for further 6 weeks. As desried ove, group A mie treted only one with PD32591 suumed to disese 8 12 weeks following esstion of drug dministrtion (Fig. 5d). By ontrst, group B mie tht reeived two yles of PD32591 remined live t time when ll of the group A mie hd een euthnized. However, ll group B mie displyed signs of melnom regrowth whih, lthough not requiring euthnsi, strongly suggested tht these mie would eventully suum to disese. Regrdless, these dt indite tht mie treted one with PD32591 to prevent melnomgenesis remin sensitive to the ntimelnom tion of seond round of PD32591 dministrtion. The results desried ove doument the potent melnomprevention tivity of PD32591 nd rpmyin. However, to determine whether these gents n eliit regression of preexisting tumors in mie, we initited melnom in group of 2 Tyr::Cre; Brf CA ; Pten lox5/lox5 mie y lol dministrtion of 4-HT to k skin (Supplementry Fig. 3). Three weeks lter, when the nimls hd redily mesurle melnom lesions, mie were rndomly divided into four groups tht were dministered either solvent ontrol (Vehile), PD32591 (PD, 12.5 mg/kg), rpmyin (Rp, 7.5 mg/kg) or the omintion of oth rpmyin nd PD32591 (PD + Rp) t the sme dose used in the single-gent rms (Fig. 6). We treted mie with these gents for 3 weeks nd mesured tumor size every dy. Melnom lesions in the vehile-treted nimls progressed stedily over the time ourse nlyzed nd hd expnded in size in ll nimls y ~4% y the end of the experiment (Fig. 6). By ontrst, mie treted with either PD32591 or rpmyin s single nture genetis volume 41 numer 5 my

5 d % Survivl Group A B Veh PD PD+relese Veh+relese Time fter Cre tivtion t = (wk) Veh PD PD/Veh tretment (6 weeks) Time fter Cre tivtion (d) t = (wk) e Bottom Middle Top % Survivl Seond PD/Veh tretment (6 weeks) Group A Group B Time fter Cre tivtion (d) Figure 5 Prevention of BRf V6E -indued melnoms y PD () Adult Tyr::CreER; Brf CA/+ ; Pten lox4-5/lox4-5 mie were treted topilly on the er with 4-HT. One week lter, mie were rndomly ssigned to e dministered PD32591 (12.5 mg/kg, n = 12) or the solvent ontrol (n = 12) for 6 weeks. Mie were nlyzed dily for the presene of utneous mlignnt melnom nd euthnized ording to stndrd ody onditioning sore. After 6 weeks of PD32591 tretment, seven drug-treted nd six ontrol mie were euthnized for nlysis of skin setions. A further five PD32591-treted nd six ontrol mie were monitored over the ourse of further 11 weeks without ny further drug dministrtion. These mie were nlyzed for the presene of utneous mlignnt melnom s desried ove. Mouse survivl ws plotted using Kpln-Meier survivl urve. () Representtive ntomil nd histologil imges of skin from 4-HT treted Tyr::CreER; Brf CA/+ ; Pten lox4-5/lox4-5 mie nlyzed fter 6 weeks of tretment with ontrol solvent (n = 6, top) or PD32591 (n = 7, middle) or from PD32591 treted mie left without further drug dministrtion for n dditionl 9-11 weeks (ottom). Sle rs: enter olumn, 2 mm; top right,.4 mm; enter nd ottom right,.1 mm. () Kpln-Meier survivl urve of vehile (n = 6) or PD32591 (n = 5) treted ohorts of mie nlyzed in this experiment. All mie were treted for the indited 6 weeks s in. Log rnk tests of survivl plots of the dt demonstrte sttistilly signifint differene etween vehile nd PD32591 treted nimls (P =.24). (d) Three week old Tyr::CreER; Brf CA/+ ; Pten lox4-5/lox4-5 mie (n = 1) were treted topilly on the right er with 4-HT. Ten dys lter ll mie were dministered PD32591 (12.5mg/kg) for 6 weeks. Drug dministrtion ws then esed for 8 weeks. Mie were then rndomized into two groups: group A (lue) ws dministered vehile ontrol nd group B (red) ws dministered PD32591 for further 6 weeks. At the end of this period mie were monitored prospetively for disese progression s desried ove. (e) Mouse survivl ws plotted using Kpln-Meier survivl urve tht demonstrted sttistilly signifint differene (P =.18) etween the groups y log rnk tests. gents hd mrked inhiition of tumor growth, ut with little or no evidene of tumor regression. Mie treted with omintion of rpmyin nd PD32591 showed ~2% redution in tumor size tht ws sttistilly signifint. These dt suggest tht the omintion of MEK1/2 nd mtor1 inhiition n led to signifint tumor shrinkge in the BRf V6E ; Pten null melnom model. Two hours fter the finl dministrtion of the vrious gents in the experiment desried ove, tumor speimens were prepred nd stined with (i) hemtoxylin nd eosin; (ii) ntiser ginst Ki67 to ssess ell prolifertion; (iii) ntiser ginst phospho-erk1/2 (perk1/2); nd (iv) ntiser ginst phospho-4ebp1 (p4ebp1) s indited (Fig. 6). The lst two stins were used to ssess the effets of PD32591 or rpmyin on the relevnt signling pthwys. Histologil nlysis of setions stined with hemtoxylin nd eosin reveled the presene of vile tumor ells in ll tretment groups, ut with n inrese in the numer of ells with pyknoti fetures of poptosis in mie treted with PD32591 or PD32591 in omintion with rpmyin (Supplementry Fig. 6 online). Administrtion of PD32591 or rpmyin, either lone or in omintion, led to redution in ell prolifertion s mesured y Ki67 stining. As expeted, tretment with PD32591 led to deresed perk1/2 stining ut hd little or no effet on p4ebp1. Conversely, tretment with rpmyin led to deresed p4ebp1 stining ut hd little or no effet on perk1/2. As expeted, the omintion of PD32591 nd rpmyin led to deresed stining of oth perk1/2 nd p4ebp1. These dt suggest tht, s single gents, PD32591 nd rpmyin hve predominntly ytostti effet on melnom initited y expression of Brf V6E nd extintion of Pten. By ontrst, evidene of melnom regression in mie treted with the omintion of PD32591 nd rpmyin suggests tht these gents my omine to eliit ytotoxi effet on preexisting lesions, supported y the derese in tumor size ompnied y n inrese in pyknoti tumor ells. However, it remins possile tht oth tumor ell utonomous nd nonutonomous effets of these gents my ontriute to melnom regression in this experimentl system. Response of BRf V6E -indued melnom ell lines to phrmologil inhiition of MEK1/2 nd mtor1 To hrterize potentil melnom ell utonomous effets of rpmyin nd PD32591, we estlished smll numer of mel- 548 volume 41 numer 5 my 29 nture genetis

6 p-4ebp1 p-erk1/2 Ki67 H&E Tumor size hnge (%) Drug or vehile dministrtion Time fter Cre tivtion t = (wk) Veh i Time (d) PD ii Rp Vehile PD PD/Rp PD+Rp nom-derived ell lines. One suh line tht grew well in ulture (2697T) ws derived from primry utneous lesion rising in 4-HT treted Tyr::CreER; Brf CA ; Pten lox5/lox5 white mouse nd ws onfirmed to hve reomined the Brf CA nd the Pten lox lleles y PCR (Supplementry Fig. 5 online). These ells showed evidene of phosphorylted MEK1/2, ERK1/2, p7 S6K nd 4EBP1, onsistent with the geneti ltertions in Brf nd Pten tht promote melnom initition nd progression in this model (Fig. 7). Tretment of melnom ells with rpmyin (25 nm) for either 24 or 48 h led to 5% inhiition of ell prolifertion fter 72 h ompred to the ontrol (Fig. 7). The ntiprolifertive effets of rpmyin were ompnied y roust inhiition of phospho-p7 S6K nd 4EBP1, even t the iii iv Figure 6 Melnom regression in response to omintion tretment with PD32591 nd rpmyin. () Melnom ws initited in group of 2 Tyr::Cre; Brf CA ; Pten lox/lox mie y lol dministrtion of 1 2 µl of 5 mm 4-HT to k skin. Three weeks lter, when the nimls hd redily mesurle melnom lesions, mie were rndomly divided into four groups tht were dministered either (i) vehile ontrol (lue); (ii) PD32591 (PD, 12.5 mg/kg, red); (iii) rpmyin (Rp, 7.5 mg/kg, green); or (iv) the omintion of oth rpmyin nd PD32591 (PD+Rp, ornge) for three weeks s indited. () Tumor size in eh of the tretment groups desried in ws mesured dily nd plotted s perentge hnge in tumor size ompred to the strting size. Two-wy ANOVA nlysis demonstrtes sttistil signifine: Vehile versus Rp or PD or PD+Rp (P <.1); PD versus PD+Rp (P =.24) nd Rp versys PD+Rp (P =.12). () Two hours following the finl dministrtion of the vrious gents, mie were euthnized nd melnom speimens were prepred for stining with hemtoxylin nd eosin or ntiser ginst Ki67, perk1/2 or p4ebp1 s indited. Sle rs,.1 mm. lowest dose tested (2.5 nm, Fig. 7d). hd no effet on the phosphoryltion of upstrem Akt (T38 or S473) nor on the phosphoryltion of MEK1/2. Tretment of 2697T ells with PD32591 (2 µm) led to omplete rogtion of ell prolifertion (Fig. 7), mrked redution in S-phse ells (Fig. 7) nd modest redution in ell numer t the ltest time point. Consistent with this, MEK1/2 inhiition eliited sustntil redution of phospho-erk1/2 with no effet on phospho-akt (Fig. 7). Moreover, MEK inhiition ws ompnied y indution of the propoptoti Bl-2 fmily protein Bim-EL, levge of spse 3 (Fig. 7) nd n inrese in ells with su-g1 DNA ontent (Fig. 7). These dt re similr to those otined in on fide humn melnom ell lines where regultion of BIM-EL expression downstrem of onogeni NRAS or BRAF ontriutes to suppression of melnom ell poptosis 3,31. Reltive ell numer Time (d) Vehile PD32591 Vehile <G1.4% G1 74.4% S 19.% G2 4.4% PD32591 <G1 12.2% G1 81.% S.5% G2 4.4% <G1.4% G1 71.4% S 22.3% G2 3.9% Figure 7 Inhiition of mouse melnom ell prolifertion y rpmyin or PD32591 in vitro. A melnom ell line (2697T) ws derived from 4-HT indued lesion tht developed on the skin of white Tyr::CreER; Brf CA/+ ; Pten lox5/lox5 mouse. As indited, 2697T ells growing synhronously in full medi were treted with PD32591 (2 µm) or rpmyin (5 nm). () Cell growth ws mesured using stndrd rystl violet stining ssy. () DNA synthesis nd ontent ws ssessed t 48 h using stndrd nti- BrdU/Propidium Iodide FACS nlysis following 2 h BrdU pulse. The perentge of ells in eh portion of the ell yle is indited. () 2697T ells, growing synhronously in full medi, were treted with 2 µm PD32591 for h t whih time ell extrts were prepred. Expression of Bim-EL, ERK1/2, Akt1, Cspse 3 nd its tive levge produt (rrowhed) s well s the phosphoryltion of ERK1/2 nd Akt1 ws ssessed y immunolotting. (d) Serum-deprived 2697T ells were pretreted with different onentrtions of rpmyin (2.5-25nM) for 3 min efore stimultion with 1% (v/v) FCS for further 2 min. Expression of Akt1, MEK1/2 nd tin s well s the phosphoryltion of 4EBP1, p7 S6K, nd Akt1 (S473 nd T38) ws ssessed y immunolotting. FITC-nti-BrdU , , , Propidium iodide PD d BimEL p-4ebp1 p-p7s6k Csp3 p-akt (S473) p-akt (T38) Akt p-erk1/2 p-mek1/2 Erk1/2 Mek Atin p-akt1 Akt nture genetis volume 41 numer 5 my

7 DISCUSSION Mouse models hve provided key insights into ner initition, progression nd therpy, nd the model desried here offers dditionl distint nd importnt dvntges in the study of melnom nd its tretment Here, we provide evidene of oopertive intertions of two signture muttions found in humn melnom, enling the genertion of mouse tht repitultes hllmrk fetures of the disese, inluding metstses to relevnt orgn sites. Moreover, the use of Brf CA mie llows urte modeling of the erliest effets of physiologil levels of expression of Brf V6E, the purported inititing muttion tht promotes the onversion of norml humn melnoytes to enign nevus ells 4,7. Beuse expression of Brf V6E promotes the development of enign melnoyti hyperplsis tht fil to progress to mlignnt melnom, this system my e used to explore mehnisms onstrining progression in vivo, to disover nd vlidte tumor suppressor genes governing suh progression hekpoints nd to define the mehnisms driving invsion nd metstsis 1,35,36. Next, use of the Tyr::CreER T2 trnsgene ffords the experimentl dvntge of exquisite sptil nd temporl ontrol over the geneti ltertions tht initite melnom formtion in mie. Finlly, nd most notly, we provide evidene tht this model is useful to study the signling xis of these key geneti lesions nd to ssess the impt of gents trgeting these pthwys tht my filitte the development of new strtegies to trget mlignnt melnom using onventionl or trgeted therpies 37. Dt presented here re onsistent with effets of trnsgeni overexpression of BRAF V6E in zerfish, whih leds to the development of fish nevi 38. Moreover, when omined with loss of Trp53 expression, BRAF V6E eliited mlignnt fish melnom. However, the relevne of this omintion of geneti events to humn melnom is unler given the reltively low frequeny of TP53 muttions in the humn disese 1,13. However, these dt stnd in ontrst to the reported effets of BRAF V6E overexpression in humn melnoytes engineered to express the tlyti suunit of telomerse (htert), dominnt-negtive TP53 R248W nd CDK4 R24C, the lst eing resistnt to the inhiitory effets of the melnom suppressor, p16 INK4A (ref. 39). In this ontext, the effets of etopi BRAF V6E expression were enign, induing only mild juntionl melnoyte nesting. By ontrst, etopi expression of onogeni vrints of RAS (NRAS G12V or HRAS G12V ) or the tlyti suunit of PI3 -kinse-α (p11-caax) led to mrked mlignnt trnsformtion of suh initited humn melnoytes. It is unler why the effet of BRAF V6E expression is so enign in this ontext, espeilly given the notle effets of BRAF V6E in zerfish nd mie. Although geneti nlysis of humn melnoms suggests tht mutted BRAF oopertes with loss of PTEN expression in melnomgenesis, it hs yet to e unequivolly demonstrted tht suh is the se 14,15. However, dt presented here demonstrte the ility of Brf V6E expression to ooperte with Pten silening in the genesis of metstti melnom. It remins to e seen whether PI3 -lipid phosphtse tivity is key to Pten s tumor suppressor tivity, espeilly in view of reent demonstrtions of effets of Pten on other signling pthwys tht re independent of its lipid phosphtse tivity 4. A key feture of this model is the ility to ontrol melnom initition nd progression in response to expression of onogeni Brf V6E in omintion with ltertions in other genes implited in melnom initition, progression or response to therpy, suh s Cdkn2, Ctnn1, Mitf or vrious lleles of M1r 5,41,42. Given the high frequeny of invsion nd metstsis oserved in this model, it my e possile to initite primry mlignnt melnom in redily essile site, reset the primry lesion nd then monitor the mie prospetively for lol or distnt reurrene. This spet of the model my fford detiled nlysis of mehnisms tht promote melnom ell migrtion, lol invsion nd metstsis in mnner tht more losely repitultes the linil ourse of melnom reurrene in humns. Metstti melnom presents one of the most diffiult diseses to tret in medil onology. Indeed, there hs not een mjor rekthrough in the tretment of this disese in over 25 yers. However, onsiderle exitement hs een generted y the identifition of geneti nd epigeneti ltertions tht ontriute to melnomgenesis, ll the more so given the elerted pe of development of phrmologil gents tht trget key signling pthwys in the melnom ell, lthough it remins unler how est to use suh gents in the lini. Studies with the rod spetrum Rf inhiitor Sorfeni, either s single gent or in omintion hemotherpy, hve proven disppointing in treting metstti melnom, ut this my e result of inomplete inhiition of BRAF V6E tivity 43,44. Moreover, individul se reports indite tht even highly ggressive melnoms expressing muttionlly tivted KIT n show mrked response to the potent -KIT inhiitor, Gleeve 27,45. Suh oservtions rise hope tht phrmologil trgeting of relevnt moleulr trget(s) my led to linil responses even in individuls with lte-stge melnom. Given the prevlene of BRAF nd PTEN ltertions in melnom, testing suh gents in vlidted prelinil mouse model my inform the linil development of vrious new nd more seletive BRAF, MEK, PI3 -kinse, AKT nd mtor inhiitors Experiments presented here suggest suh gents my hve vlue in melnom prevention nd therpy, ut the relpse of mie following esstion of drug dministrtion indites tht single-gent therpy with MEK1/2 or mtor inhiitors is unlikely to ure individuls with this disese. However, this model system is redily trtle to test omintions of trgeted gents or trgeted therpy omined with onventionl ytotoxis or other gents suh s iohemotherpy or immune-sed pprohes 49. Suh studies might provide useful guides to the est dosge nd sheduling of ntimelnom therpeutis in linil trils in humns. METHODS Mouse reeding, tivtion of Tyr::CreER trnsgene nd tretment of mie with trgeted therpeutis. Brf CA, Tyr::CreER nd Pten lox4-5 mie were genotyped s previously desried 17,2,24. Cre-medited onversion of Brf CA to Brf VE nd the deletion of exons 4 nd 5 of Pten were ssessed y PCR s previously desried. Topil dministrtion of 4-hydroxytmoxifen (4-HT) ws onduted y prepring 25 5 mg/ml (65 13 mm) solution of 4-HT (7% Z-isomer, Sigm) in dimethylsulphoxide nd pplying enough solution to wet the right er, right flnk nd til with smll pint rush on postntl dys 2, 3 nd 4. For lolized melnom indution on the k skin, dult (6 8 weeks of ge) mie were treted topilly with 1 2 µl of 1.9 mg/ml (5 mm) 4-HT t 6 8 weeks of ge using similr protool. Generlized indution in dult mie ws performed y intrperitonel injetion of 1 mg of tmoxifen per 4 g mouse on three onseutive dys. In this se tmoxifen ws prepred s 1 mg/ml suspension in penut oil. PD35291 ws dissolved in.5% (w/v) hydroxy-propyl-methylellulose,.2% (v/v) Tween 8 (Sigm) nd dministered to mie dily y orl gvge t dose of 12.5 mg/ kg. (LC Lortories) ws suspended in.5% (w/v) methylellulose nd dministered to mie dily y orl gvge t dose of 7.5 mg/kg. The relevnt solvent ws dministered to ontrol nimls in the melnom prevention studies. Tissues were prepred for nlysis s previously desried 17,2. Genertion of mie rrying Cre-intivted llele of Pten. A trgeting vetor ws generted in pko SeletDT-915 using 12-k frgment of the mouse Pten gene y stndrd tehniques. A ssette rrying Pten exon 5 flnked y loxp sites nd PGK::Neo flnked y FRT sites ws then suloned etween the genomi DNA rms. This onstrut ws linerized, trnsfeted into TC1 ES ells nd genomi DNA from G418 nd diphtheri toxin resistnt ES ells were sreened y Southern lot nd PCR for homologous reomintion events. Blstoyst injetions were rried out with three different trgeted lones, nd trnsmit- 55 volume 41 numer 5 my 29 nture genetis

8 ting himeri mie were red from CAGG Flpe trnsgeni mie to generte the Pten lox5 llele 5. Chimeri mie were mted with FVB/N mie nd trnsmission verified y Southern lotting, nd PCR with the primers listed in Supplementry Tle 2 online. All mie were mintined on mixed C57Bl/6 nd FVB/N kground y rndom interreeding. Melnom ell line derivtion nd ulture. Melnom ell lines were derived y physil resetion of primry utneous lesions followed y triturtion through 2-guge syringe needle nd filtrtion through 1 µm nylon ell striner (BD/ Flon) efore eing pled into medi. Cell suspensions were plted in medi ( 1:1 mix of DMEM nd Hms F12) supplemented with FCS (5% v/v) nd peniillin, streptomyin nd glutmine. Serum strved ells were treted with different doses of rpmyin ( nm s indited) for 3 min efore 1%(v/v) serum stimultion for n dditionl 2 min. Asynhronously growing ells in full medi were treted with PD32591 (2 µm) for different periods of time (24 96 h s indited) with ell prolifertion quntified y stining of fixed ells with rystl violet. Dt presented in Figure 6 re results from two seprte experiments in whih eh time point ws mesured in triplite. Suonfluent ells grown in full melnoyte medi treted with PD32591 (2uM) or rpmyin (5nM) were ultured for 46 h when BrdU ws dded to 2uM for 2 h. Single ell suspensions fixed in ethnol were stined with FITC-leled BrdU (BD Phrmingen 55628), inuted with propidium iodide (1 µg/ml), nd nlyzed with BD FACS lier. Cell extrts were prepred y stndrd tehniques nd resolved using SDS-polyrymide gel eletrophoresis. Protein lots prepred y stndrd tehniques, nd were proed with ommerilly ville ntiser ginst vrious proteins s indited 31. Immunohemistry nd immunolotting. The expression of tyrosinse (Tyr, Alino), tyrosinse-relted protein 1 (Tyrp1, Brown) or tyrosinse-relted protein 2 (Tyrp2, Slty) ws deteted y stining with ntiser ginst αpep7, αpep1 or αpep8, respetively (gift of V. Hering, Ntionl Cner Institute) 26. Slides were developed using di-mino-enzidine-peroxidse sustrte kit (Vetor Ls), ording to the mnufturer s instrutions. The expression nd phosphoryltion of proteins ws deteted y immunolotting using pproprite ommerilly ville ntiser: phospho-4ebp1, phospho-p7 S6K, phospho-mek1/2, totl MEK1/2, phospho-erk1/2; totl ERK1/2; phospho S473-AKT; phospho- T38-AKT; totl AKT; spse 3 (ll from Cell Signling Tehnology), Bim-EL (Cliohem ma 14A8); nd tin (Sigm). Note: Supplementry informtion is ville on the Nture Genetis wesite. AUTHOR CONTRIBUTIONS M.M. nd M.B. estlished ollortion for the exhnge of relevnt mouse strins to enle the genertion nd nlysis of Tyr::CreER, Brf CA, Pten lox mie nd ontriuted to the mnusript eqully s senior uthors. D.D. (UCSF) nd D.P.C. (University of Vermont) performed ll of the mouse experiments in prllel. Representtive figures were seleted for pulition y D.D., D.P.C., M.M. nd M.B. nd were prepred for pulition y D.D. R.A.C. performed immunolot nlysis of 2697T ells treted with PD B.N. nd W.E.D. ssisted with nd optimized mouse tumor indution nd performed immunohistohemil nlysis. A.N.K. nlyzed Tyr::CreER, Brf CA, Pten lox4-5 mie treted with multiple yles of PD M.J.Y. generted nd hrterized Pten lox5 mie in the lortory of R.A.D. M.M. wrote the mnusript nd shepherded it through review with ontriutions from D.D., D.P.C., M.J.Y., R.A.D. nd M.B. ACKNOWLEDGMENTS We thnk the memers of the MMhon nd Bosenerg lortories s well s B. Bstin, L. Chin, E. Filenov, B. Hnn, M. Herlyn, L. Johnson, G. Merlino, P. P. Pndolfi, V. Hering, M. Held, G. Ky nd D. Mtzen for the provision of mouse strins, regents, dvie nd support. M.M. thnks A. Rirt nd J. Seolt- Leopold (Pfizer) for provision of PD32591 nd knowledges the support of the University of Cliforni Sn Frniso Helen Diller Fmily Comprehensive Cner Center Mouse Pthology nd Pre-Clinil Therpeutis ores. R.A.D. is supported s n Amerin Cner Soiety Reserh Professor. This work ws supported y grnts from the Melnom Reserh Foundtion, U.C. Disovery Awrd nd from the US Ntionl Institutes of Helth (CA to M.M., CA to R.A.D. nd CA nd CA to M.B., respetively). Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/ 1. Chin, L., Merlino, G. & DePinho, R.A. Mlignnt melnom: modern lk plgue nd geneti lk ox. Genes Dev. 12, (1998). 2. Gry-Shopfer, V.C., d Roh Dis, S. & Mris, R. The role of B-RAF in melnom. Cner Metstsis Rev. 24, (25). 3. Chin, L. The genetis of mlignnt melnom: lessons from mouse nd mn. Nt. Rev. Cner 3, (23). 4. Dvies, H. et l. Muttions of the BRAF gene in humn ner. Nture 417, (22). 5. Grrwy, L.A. et l. Integrtive genomi nlyses identify MITF s linege survivl onogene mplified in mlignnt melnom. Nture 436, (25). 6. Hywrd, N.K. Genetis of melnom predisposition. Onogene 22, (23). 7. Pollok, P.M. et l. High frequeny of BRAF muttions in nevi. Nt. Genet. 33, 19 2 (23). 8. Wellrok, C., Krsrides, M. & Mris, R. The RAF proteins tke entre stge. Nt. Rev. Mol. Cell Biol. 5, (24). 9. Merer, K.E. & Prithrd, C.A. Rf proteins nd ner: B-Rf is identified s muttionl trget. Biohim. Biophys. At 1653, 25 4 (23). 1. Mihloglou, C. et l. BRAFE6-ssoited senesene-like ell yle rrest of humn nevi. Nture 436, (25). 11. Svidersky, E.V. et l. p16(ink4) in melnoyte senesene nd differentition. J. Ntl. Cner Inst. 94, (22). 12. Wellrok, C. et l. V599EB-RAF is n onogene in melnoytes. Cner Res. 64, (24). 13. Chin, L., Grrwy, L.A. & Fisher, D.E. Mlignnt melnom: genetis nd therpeutis in the genomi er. Genes Dev. 2, (26). 14. Lin, W.M. et l. Modeling genomi diversity nd tumor dependeny in mlignnt melnom. Cner Res. 68, (28). 15. Tso, H., Goel, V., Wu, H., Yng, G. & Hlusk, F.G. Geneti intertion etween NRAS nd BRAF muttions nd PTEN/MMAC1 intivtion in melnom. J. Invest. Dermtol. 122, (24). 16. Tso, H., Zhng, X., Fowlkes, K. & Hlusk, F.G. Reltive reiproity of NRAS nd PTEN/MMAC1 ltertions in utneous melnom ell lines. Cner Res. 6, (2). 17. Dnkort, D. et l. A new mouse model to explore the initition, progression, nd therpy of BRAFV6E-indued lung tumors. Genes Dev. 21, (27). 18. Tonks, I.D. et l. Tyrosinse-Cre mie for tissue-speifi gene ltion in neurl rest nd neuroepithelil-derived tissues. Genesis 37, (23). 19. Guyonneu, L., Murisier, F., Rossier, A., Moulin, A. & Beermnn, F. Melnoytes nd pigmenttion re ffeted in dophrome tutomerse knokout mie. Mol. Cell. Biol. 24, (24). 2. Bosenerg, M. et l. Chrteriztion of melnoyte-speifi induile Cre reominse trnsgeni mie. Genesis 44, (26). 21. Bennett, D.C. Humn melnoyte senesene nd melnom suseptiility genes. Onogene 22, (23). 22. Jonsson, G. et l. Genomi profiling of mlignnt melnom using tiling-resolution rrycgh. Onogene 26, (27). 23. Willmore-Pyne, C., Holden, J.A., Hirshowitz, S. & Lyfield, L.J. BRAF nd -kit gene opy numer in muttion-positive mlignnt melnom. Hum. Pthol. 37, (26). 24. Trotmn, L.C. et l. Pten dose dittes ner progression in the prostte. PLoS Biol. 1, E59 (23). 25. Inoue-Nrit, T. et l. Pten defiieny in melnoytes results in resistne to hir grying nd suseptiility to rinogen-indued melnomgenesis. Cner Res. 68, (28). 26. Jimenez, M., Tsukmoto, K. & Hering, V.J. Tyrosinses from two different loi re expressed y norml nd y trnsformed melnoytes. J. Biol. Chem. 266, (1991). 27. Hodi, F.S. et l. Mjor response to imtini mesylte in KIT-mutted melnom. J. Clin. Onol. 26, (28). 28. Ohren, J.F. et l. Strutures of humn MAP kinse kinse 1 (MEK1) nd MEK2 desrie novel nonompetitive kinse inhiition. Nt. Strut. Mol. Biol. 11, (24). 29. Stini, D.M., Erdjument-Bromge, H., Lui, M., Tempst, P. & Snyder, S.H. RAFT1: mmmlin protein tht inds to FKBP12 in rpmyin-dependent fshion nd is homologous to yest TORs. Cell 78, (1994). 3. Sheridn, C., Brumtti, G. & Mrtin, S.J. Onogeni B-RfV6E inhiits poptosis nd promotes ERK-dependent intivtion of Bd nd Bim. J. Biol. Chem. 283, (28). 31. Crtlidge, R.A. et l. Onogeni BRAF(V6E) inhiits BIM expression to promote melnom ell survivl. Pigment Cell Melnom Res. 21, (28). 32. Wlker, G.J. & Hywrd, N.K. Pthwys to melnom development: lessons from the mouse. J. Invest. Dermtol. 119, (22). 33. Brdeesy, N., Wong, K.K., DePinho, R.A. & Chin, L. Animl models of melnom: reent dvnes nd future prospets. Adv. Cner Res. 79, (2). 34. Tietze, M.K. & Chin, L. Murine models of mlignnt melnom. Mol. Med. Tody 6, (2). 35. Woods, D. et l. Rf-indued prolifertion or ell yle rrest is determined y the level of Rf tivity with rrest medited y p21cip1. Mol. Cell. Biol. 17, (1997). 36. Zhu, J., Woods, D., MMhon, M. & Bishop, J.M. Senesene of humn firolsts indued y onogeni Rf. Genes Dev. 12, (1998). 37. Olive, K.P. & Tuveson, D.A. The use of trgeted mouse models for prelinil testing of novel ner therpeutis. Clin. Cner Res. 12, (26). nture genetis volume 41 numer 5 my

9 38. Ptton, E.E. et l. BRAF muttions re suffiient to promote nevi formtion nd ooperte with p53 in the genesis of melnom. Curr. Biol. 15, (25). 39. Chudnovsky, Y., Adms, A.E., Roins, P.B., Lin, Q. & Khvri, P.A. Use of humn tissue to ssess the onogeni tivity of melnom-ssoited muttions. Nt. Genet. 37, (25). 4. Freemn, D.J. et l. PTEN tumor suppressor regultes p53 protein levels nd tivity through phosphtse-dependent nd -independent mehnisms. Cner Cell 3, (23). 41. Lrue, L. & Delms, V. The WNT/Bet-tenin pthwy in melnom. Front. Biosi. 11, (26). 42. Lndi, M.T. et l. MC1R germline vrints onfer risk for BRAF-mutnt melnom. Siene 313, (26). 43. Eisen, T. et l. Sorfeni in dvned melnom: Phse II rndomised disontinution tril nlysis. Br. J. Cner 95, (26). 44. Flherty, K.T. Chemotherpy nd trgeted therpy omintions in dvned melnom. Clin. Cner Res. 12, 2366s 237s (26). 45. Lutzky, J., Buer, J. & Bstin, B.C. Dose-dependent, omplete response to imtini of metstti muosl melnom with K642E KIT muttion. Pigment Cell Melnom Res 21, (28). 46. Mir, S.M. et l. Identifition nd hrteriztion of NVP-BEZ235, new orlly ville dul phosphtidylinositol 3-kinse/mmmlin trget of rpmyin inhiitor with potent in vivo ntitumor tivity. Mol. Cner Ther. 7, (28). 47. Tsi, J. et l. Disovery of seletive inhiitor of onogeni B-Rf kinse with potent ntimelnom tivity. Pro. Ntl. Ad. Si. USA 15, (28). 48. Yeh, T.C. et l. Biologil hrteriztion of ARRY (AZD6244), potent, highly seletive mitogen-tivted protein kinse kinse 1/2 inhiitor. Clin. Cner Res. 13, (27). 49. Sosmn, J.A. & Puznov, I. Moleulr trgets in melnom from ngiogenesis to poptosis. Clin. Cner Res. 12, 2376s 2383s (26). 5. Rodriguez, C.I. et l. High-effiieny deleter mie show tht FLPe is n lterntive to Cre-loxP. Nt. Genet. 25, (2). 552 volume 41 numer 5 my 29 nture genetis