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EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

A Mix of Science and Se r endipity: PDGF Receptor–Driven Leukemias and Sa r coma

nan nan

A t h i rd e xa m p l e co m es fr o m d ermat o fi b r o sarc o ma p r o t ub era n s , a sarc oma c h a r acteri zed by a t( 17,22 ) tra n sl o cati on t h at f u ses t h e COL 1 A g e n e t o t he P DG FB li gand ( no t t he r ecep t o r) . COL 1 A-PDGFB is on c og e n ic t h r ough a u t o c r i ne s tim u l a ti on o f t he no rmal PDGF rece p t o r i n t h ese t u m o r cells .

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

A Mix of Science and Se r endipity: PDGF Receptor–Driven Leukemias and Sa r coma

nan nan

Patie n ts w it h de rm a t o fi b r osa rc o ma p r o t ub era n s res pond t o imati n i b t h era py b eca u se it t a r ge t s t he P DG F rece p t o r , j u st on e ste p do w n stream fr o m t h e on c og e nic l es i on .

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Exploiting the New Paradigm: Sea r ching for Other Kinase-Driven Cancers

nan nan

Th e b e ne fit s o f se r end i p it y no twit h sta nd i ng, t h e g r o wi ng nu m b er o f e x am p les o f success f u l k i nas e i nh i b it o r t h era py i n t u m o rs wit h a m u tati on or am p lifi ca ti on o f t he d r ug t a r g et b e gg e d f o r a m o re rati on al a pp r o ac h t o drug d is cove r y and deve l op me n t . I n 2002, t h e list o f hu ma n t u m o rs known t o h a v e mu t a ti ons i n k i nases w as qu ite small . D u e t o a dv a n ces i n a u t o ma ted g e n e se quenc i ng, it beca m e po ssi b le t o as k w h et h er a m u c h la r g er fracti on of hu ma n cance r s mi gh t a l so h a v e s u c h m u tati on s t h r ough a b r u te f o rce a ppro ach . T o add r ess t h i s que sti on c o m p re h e n si v el y , on e w ou l d h a v e t o se qu e n c e a ll o f t he k i nases i n t h e g e no me i n hund re d s o f sam p les o f eac h t u m or ty pe. S eve r a l ea rl y p il ot st ud ies d em on strate d t h e po te n tial o f t h is a ppro ach by r evea li ng im po rta n t n ew ta r g ets f o r d r ug d e v el op me n t . Perh aps t h e m o s t spec t acu l a r was t he disc ov er y o f m u tati on s i n t h e BRAF k i n ase in ov e r h alf o f pa ti en t s w it h m e l ano ma , as well as i n a smaller fracti on o f c o l on a nd t hy r o i d cance r s . A no t h er was t h e d isc ov er y o f m u tati on s i n t he J AK2 k in ase i n nea rl y a ll pa ti en ts wit h po l y c y t h emia v era , as well as a si gn i f ic an t fr ac ti on o f pa ti en ts wit h m y el o fi b r o sis a nd esse n tial t hro m bocy t os i s . A t h ir d ex am p le was t h e i d e n tificati on o f PIK 3 CA m u tati ons i n a va ri e t y o f t u m o rs , wit h t h e g reatest fre qu e n cies i n b reast , e ndo me t ri a l , and co l o r ec t a l c a n cers . PIK 3 CA e n c od es a li p i d k i n ase t hat g e n e r ate s t he second m esseng er pho s ph ati dy l i no sit o l 3 - pho s ph ate (PIP 3 ) . P IP3 acti va t es g r ow t h and su r v i v al si gn ali ng t h r ough t h e AKT famil y o f k i n ases a s we ll as o t he r downs tream e f fect o rs . C oup le d wit h t h e well-esta b lis hed r o l e o f t he phosph atase a nd te n si n ho m o l og (PTEN) li p i d pho s ph at ase i n dephospho r y l a ti ng PIP 3, t h e d isc ov er y o f PIK 3 CA m u tati ons f ocused tr e m endou s atte n ti on on d e v el op i ng i nh i b it o rs at

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Exploiting the New Paradigm: Sea r ching for Other Kinase-Driven Cancers

nan nan

m u lti p le l eve l s o f t h i s pa t hwa y , as d isc u sse d f u rt h er i n t h e f o ll o w p a r a gr a phs. E ac h o f t hese im po rt an t d i s c ov eries — BRA F , JAK 2, a nd PIK 3 CA — came fro m r e l a ti ve l y s m a ll e f f o rts (less t h a n 100 t u m o rs) a nd g e n erall y fo c u se d o n r esequenc i ng on l y t ho se e xon s t h at c od e d f o r re g i on s o f k i n a ses wh e r e mu t a ti ons had been f ound i n o t h er k i n ases (t yp icall y , t h e j ux tamem b r ane and k i nase do mai n s) . T h ese restricte d searc h es were la r gely dr i v e n by t he h i gh cos t o f D NA se qu e n ci ng u si ng t h e Sa ng er met hod. I n 2006, a co m p r ehens i ve e f f o rt t o se qu e n ce all o f t h e e xon s i n all k i n ases i n 100 t u m o r s cou l d eas il y exce e d se v eral milli on do llars . Fi n a n cial s uppo rt for s u c h p r o j ec t s cou l d no t be ob tai n e d easil y t h r ough tra d iti on al f und i ng a g e n cie s because t he ri sk /r ewa r d was c on si d ere d t oo h i gh. F u rt h erm o re , s ub sta n ti a l i n fr as tr uc t u r e f o r sam p le ac qu isiti on, micr od issecti on o f t h e t u m or s f r o m no rm a l ti ssue, nu cleic aci d p re p arati on, h i gh t h r oughpu t a u t o mat ed sequenc i ng, and co m pu tati on al a n al y sis o f t h e res u lti ng d ata was esse n tia l. F ew i ns tit u ti ons wer e e qu i pp e d t o a dd ress t h ese c h alle ng es . I n r es pon s e, t he Na ti ona l Cance r I n stit u te i n t h e U n ite d States (i n p art n ers hip w it h t h e Na ti ona l Hu m an Geno me Researc h I n stit u te) a nd a n i n ter n ati onal group k n own as t he I n t e r na tio n al Ca n cer Ge no me C on s o rti u m (ICGC) la un c h ed l a r ge - sca l e e f f o rt s t o se qu e n ce t h e c o m p lete g e no mes o f t hou sa nds of ca n cer s. I n pa r a ll e l , nex t- gen erati on se qu e n ci ng tec hno l og ies res u lte d in massi v e r educ ti ons i n cos t , a l lo wi ng a m o re c o m p re h e n si v e a n al y sis o f m u c h la r ge r nu m be r s o f t u m or s . At t h e time o f t h is writi ng, t h e US eff o rt ( calle d T he Cance r Geno m e A tlas [TCGA]) h a d re po rte d d ata on 29 d i f f e r e n t t u m o r t ypes ( ) . T h e i n ter n ati onal c on s or ti u m has co mmitt ed t o s e qu e n ci ng 25,000 t u m o rs re p rese n ti ng 50 d i f f e r e n t cance r sub t ypes B ot h g r oup s h a v e e n f o rce d imme d iate release o f all se quence i n f o rm a ti on t o t he researc h c o mm un it y free o f c h a r g e s o t hat t h e e n ti re sc i en tifi c co mm un it y ca n lear n fr o m t h e d ata . T h is po lic y e n a bled pan c ancer m u t a ti ona l ana l yse s t h at g i v e a n ov erall v iew o f t h e g e no mic la nd sca pe o f cance r , se r v i ng as a b l u e p ri n t f o r t h e c o mm un it y o f ca n cer r esea r c he r s and d r ug deve l op ers .

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Rounding Out the T r eatment of Myelop r oliferative Disorders: JAK2 and Myelofib r osis

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Rounding Out the T r eatment of Myelop r oliferative Disorders: JAK2 and Myelofib r osis

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Rounding Out the T r eatment of Myelop r oliferative Disorders: JAK2 and Myelofib r osis

nan nan

T a k e n t oge t he r w it h t he BCR- ABL tra n sl o cati on i n CML a nd FIP 1 L 1 -P DG FR- α i n H ES , t he d i scov er y o f JAK 2 m u tati on s i n po l y c y t h emia , esse n tia l t h r o m bocy t os i s, and m y el o fi b r o sis p r ov i d e d a un if y i ng und e r sta nd i ng o f m ye l op r o lif e rati v e d is o r d ers as d iseases o f a bno rmal k i n ase a c ti va ti on. T he JAK f am il y k i n ases are t h e p rimar y e f fect o rs o f si gn ali ng t h r ough i n fl a mm a t ory c y t ok i n e rece p t o rs a nd, t h eref o re , h a d been c on si d e red co m pe lli ng t a r ge ts f o r a n ti-i n flammat o r y d r ug s . B u t t h e JAK 2 m u tati on d i scove r y imm ed i a tel y s h ifte d t h ese eff o rts t o war d d e v el op i ng J AK2 i n hi b it o r s f o r m ye l op r o liferati v e d is o r d ers . Beca u se m o st p atie n ts h a v e a c o mm on JAK2 V617F m u tati on, t h ese e f f o rts c ou l d ra p i d l y f o c us on sc r ee n i ng f o r ac ti v it y aga i ns t a si ng le g e no t yp e . Pr og ress h as b ee n ra p i d . M y el of i b r os i s was se l ec t ed a s t h e i n itial i nd icati on (i n stea d o f esse n tial t hro m bocy t os i s o r po l ycy t he mia v era) b eca u se t h e time t o re g istrati on is e xp ecte d t o be t he sho rt es t . Cu rre n tl y , r uxo liti n i b is a pp r ov e d f o r m y el of i b r os i s based on sh ri n ka g e i n s p lee n size as t h e p rimar y e ndpo i n t . Cli n ical tri a l s i n essen ti a l t h rom bo c y t o sis a nd po l y c y t h emia v era ( v ers us hydroxy ur ea ) a r e ongo i ng. O t h er JAK 2 i nh i b it o rs are als o i n cli n ical d e v el opmen t .

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

BRAF Mutant Melanoma: Several Missteps Before Finding the Right Inhibitor

nan nan

BRAF Mutant Melanoma: Several Missteps Before Finding the Right Inhibitor

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

BRAF Mutant Melanoma: Several Missteps Before Finding the Right Inhibitor

nan nan

A s w it h JAK2 m u t a ti ons i n my el op r o liferati v e d is o r d ers , t h e d isc ov er y o f BR A F mu t a ti ons i n pa ti en t s w it h mela no ma la un c h e d wi d es p rea d e f f o rt s to f i nd po te n t BRA F i nh i b it o r s. O n e earl y ca nd i d ate was t h e d r ug s o rafe n i b, wh ic h h a d been op timi zed du ri ng d r ug d isc ov er y t o i nh i b it RAF k i n ases . (Sor a f eni b a l so i nh i b it s vascu lar e ndo t h elial g r o wt h fact o r (VEGF) r ece p t ors, wh i ch l ed t o it s app r ov al i n k i dn e y ca n ce r , as d isc u sse d later i n t h is c h a p t e r . ) Desp it e t he co m p elli ng m o lec u lar rati on ale f o r tar g eti ng BR A F , c li n i ca l r esu lt s o f so r af e n i b i n mela no ma were e x tremel y

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

BRAF Mutant Melanoma: Several Missteps Before Finding the Right Inhibitor

nan nan

d isa ppo i n ti ng and r educed en t hu siasm f o r pu rs u i ng BRAF as a d r ug tar get.

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

BRAF Mutant Melanoma: Several Missteps Before Finding the Right Inhibitor

nan nan

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

BRAF Mutant Melanoma: Several Missteps Before Finding the Right Inhibitor

nan nan

In h i nd si gh t , t h i s conce r n wa s c o m p letel y mis gu i d e d. S o rafe n i b do si ng i s limite d by t ox i c iti es t ha t p r eclud e ac h ie v i ng ser u m le v els i n p atie n ts t h at po te n tl y i nh i b it RA F , bu t a r e s u f ficie n t t o i nh i b it VEGF rece p t o rs . I n a dd iti on, pa ti en t s we r e en r o lle d wit hou t scree n i ng f o r BRAF m u tati on s i n t h ei r t umo r s. A lt hough t he fre qu e n c y o f BRAF m u tati on s i n mela no ma i s h i gh, t he i nc l us i on o f pa ti en t s wit hou t t h e BRAF m u tati on d il u te d t h e c h a n ce o f see i ng any c li n i ca l si gn al . I n s ho rt , t h e cli n ical e v al u ati on o f s or a f e n i b i n m e l ano m a was poo rl y d esi gn e d t o test t h e hypo t h esis t h at BR A F is a t he r apeu ti c t a r ge t . T h e d a ng er is t h at n e g ati v e d ata fr o m s u c h cli n ical e xpe rim en t s can s l ow s ub se qu e n t p r og ress . It is critical t o kno w t he ph a r ma codyna mi c p r ope rti es o f t h e d r ug a nd t h e m o lec u lar ph e no t yp e o f t h e p ati en t s be i ng s t ud i ed wh e n i n ter p reti ng t h e res u lts o f a n e g ati v e st ud y . Th e f a c t t ha t RA F k i nases a re i n terme d iate c o m pon e n ts o f t h e well-c h a r acteri zed RA S/ mit ogen -acti v ate d p r o tei n (MAP) k i n ase p at h wa y ( t r a n s duc i ng s i gna l s fr o m RA S t o RAF t o MEK t o ERK) raise d t h e po ssi b ili ty t ha t t u m o r s w it h BR AF m u tati on s mi gh t res pond t o i nh i b it o r s o f on e of t hese downs tr ea m k i na ses ( ) . Precli n ical st ud ies re v eale d t h at t u m o r ce ll li nes w it h BR AF m u tati on were e xqu isitel y se n siti v e t o i nh i b it ors o f t he downs tr ea m k i n ase MEK . (S o rafe n i b, i n c on trast , do e s no t s ho w t h i s p r o fil e o f ac ti v it y . T hu s , p r op er p recli n ical scree n i ng w ould h a v e r ev ea l ed t he sho rt co mi ng s o f s o rafe n i b as a BRAF i nh i b it o r . ) C ur i ou sl y , ce ll li nes w it h a m u tati on o r am p lificati on o f EGFR o r HER 2, wh ic h func ti on ups tr ea m i n t h e p at h wa y , were i n se n siti v e t o MEK i nh i b iti o n . E ven t u m o r li nes w it h RAS m u tati on s were v aria b l y se n siti ve. I n s hor t , t he p r ec li n i ca l da t a m a de a str ong case t h at MEK i nh i b it o rs s hou ld be e f f ecti ve i n BRA F m u t an t m e la no ma , bu t no t i n o t h er s ub t yp es . T h e reas on t h at HER2, E G F R, and RA S m u ta n t t u m o rs were no t se n siti v e t o MEK i nh i b it ors i s exp l a i ned, a t l eas t i n p art , by t h e e x iste n ce o f n e g ati v e f ee db ac k l oops t ha t m odu l a t e t h e fl ux o f si gn al tra n s du cti on t h r ough M EK

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

BRAF Mutant Melanoma: Several Missteps Before Finding the Right Inhibitor

nan nan

In p ar a ll e l w it h t he gene r a ti on o f t h ese p recli n ical fi nd i ng s , cli n ical tr ials of se v e r al M E K i nh i b it o r s wer e i n itiate d. Patie n ts wit h v ari ou s ca n cers we r e e nro lle d i n t he ea rl y s t ud i es, bu t t h ere was a str ong b ias t o i n cl ud e mela no m a pa ti en t s. Si gn ifi can t eff o rts were ma d e t o d em on strate MEK i nh i b iti o n i n t u m o r ce ll s by m e as u ri ng t h e pho s pho r y lati on stat u s o f t h e d i r ect downs tr ea m subs tr a t e ERK u si ng a n imm unoh ist o c h emical a n al y s is of b i op si es fr o m pa ti en t s w it h metastatic d isease . P h ase I st ud ies o f t h e t wo ea r liest co m pounds i n c li n i ca l d e v el op me n t (PD 325901 a nd AZD 6244 ) do c u me n t ed r educed phospho -ERK stai n i ng at m u lti p le do se le v els i n se v e r al pa ti en t s f o r who m base li n e a nd treatme n t b i op sies were ob tai n e d. (I n t he f o ll ow i ng, we will lear n t h at t h ese ph armac odyn ami c st ud ies , wh il e we ll i n t en ti oned, were no t qu a n titati v e e nough t o do c u me nt t h e ma gn it ude o f M E K i nh i b iti on i n t h ese p atie n ts . ) F u rt h erm o re , cli n ic al r es pon s es we r e obse r ved i n a few p atie n ts wit h BRAF m u ta n t mela no m a. Ar me d w it h t h i s con fi dence, a ra ndo mize d ph ase II cli n ical trial o f AZD6244 was conduc t ed i n a dv a n ce d mela no ma , wit h t h e

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

BRAF Mutant Melanoma: Several Missteps Before Finding the Right Inhibitor

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c h em o t he r apeu ti c agen t t e m o z o l o mi d e (w h ic h is a pp r ov e d f o r g li ob las to m a ) as t he co m pa r ato r arm . (T h e cli n ical d e v el op me n t o f P D325901 was d i scon ti nued b eca u se o f safet y c on cer n s a bou t o c u lar a nd n e uro l og i c t ox i c it y . ) D i sappo i n ti ng l y , p atie n ts recei v i ng AZD 6244 h a d no b e n e f it in p r og r ess i on -fr ee su r v i v al w h e n c o m p are d t o tem o z o l o mi d e-t r eate d pa ti en t s, r a i s i ng f u rt he r c on cer n s a bou t t h e v ia b ilit y o f BRAF as a drug ta r ge t A c l ose r exa mi n ati on o f t h e d ata re v eale d t h at cli n ical r es pon s es we r e, i ndeed, seen i n p atie n ts recei v i ng AZD 6244. T h e fact t hat BR A F mu t a ti on s t a t us was n ot re qu ire d f o r st udy e n tr y li k el y d imi n is h e d t h e cli n i ca l s i gna l i n t he A Z D 6 244 arm , a less on lear n e d fr o m t h e EGFR i nh i b it or tri a l s i n l ung cance r . I nd ee d, a d i f fere n t MEK i nh i b it o r , trameti nib, r ecei v e d F DA app r ova l i n 2013 b ase d on acti v it y i n mela no ma p atie n ts with t h e BR A F m u t a ti on A ll doub t s abou t BRA F as a ta r g et v a n is h e d i n 2009 t o 2010 w h e n dr amati c c li n i ca l r esponses we re ob ser v e d wit h a nov el BRAF i nh i b it o r v em ur a fen i b (PL X4032 ) . Li ke s o rafe n i b, t h is c o m pound was op timize d t o i nh i b it R A F , bu t w it h an add iti on al f o c u s on m u ta n t BRA F . V em u rafe n ib d i f f e r s d r a m a ti ca ll y fr o m so rafe n i b b eca u se it po te n tl y i nh i b its BRAF w it hou t t he add iti ona l b r oad ra ng e o f acti v ities t h at s o rafe n i b h as a g ai n s t o t h e r k i nases li ke t he V E G F rece p t o r . T h e g reater selecti v it y o f v em ur a fen i b r e l a ti ve t o so r a f en i b res u lte d i n a m u c h g reater t o lera b ilit y , s u c h t h at it cou l d be g i ven a t hi gh do ses w h ile a vo i d i ng si gn ifica n t t ox ici t y . Th e ea r l y days o f ve m u r a f en i b cli n ical d e v el op me n t were p la gu e d by c h alle ng e s i n m ax imi z i ng t he o ral b i o a v aila b ilit y o f t h e d r ug .

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

BRAF Mutant Melanoma: Several Missteps Before Finding the Right Inhibitor

nan nan

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

BRAF Mutant Melanoma: Several Missteps Before Finding the Right Inhibitor

nan nan

C on se quen tl y , t he i n iti a l phase I cli n ical trial was tem po raril y h alte d t o d e v el op a nove l f o rm u l a ti on (i . e ., t h e c o i ng re d ie n ts i n t h e d r ug ca p s u le o r ta b let t ha t im p r ove so l ub ilit y and a b s o r p ti on t h r ough t h e g astr o i n testi n al t r act ). M uch h i ghe r se r u m l eve ls were ob tai n e d i n p atie n ts w ho recei v e d the n e w v e mu r a f en i b f o rm a ti on and, s ho rtl y t h ereafte r , c o m p lete a nd p artial r es pon s es we r e obse r ved i n abou t 80 % o f t h e mela no ma p atie n ts wit h B-R AF m u t an t t u m o r s. Stri k i ng l y , no acti v it y was ob ser v e d i n p atie n ts w hose t u m or s we r e w il d t ype f o r B RA F T h e d ata were s o c o m p elli ng t h at v em ur a fen i b was imm ed i a t e l y a dv a n ce d t o a ph ase III re g istrati on trial .

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

BRAF Mutant Melanoma: Several Missteps Before Finding the Right Inhibitor

nan nan

Simila r l y im p r ess i ve r esponse s i n BRAF m u ta n t mela no ma p atie n ts wer e ob se rv e d w it h a second po t en t RAF i nh i b it o r d a b rafe n i b , p r ov i d i ng f u rt he r proof t ha t BRA F i s a im po rt a nt ca n cer tar g et . Th e ve m u r a f en i b and dab rafe n i b d ata als o p r ov i d e i n si gh t i n t o w hy s or a f e n i b and t he ea rl y M E K i nh i b it o r trials faile d t o d em on strate acti v it y . On e les son i s t he c riti ca l im po rta n ce o f ac h ie v i ng a d e qu ate ta r g et i nh i b it ion. Cli n ical r esponses w it h ve m ur afe n i b were ob ser v e d on l y after t h e d r ug was r e for m u l a t ed t o ach i eve subs t an tiall y h i gh er ser u m le v els . Re du cti on s i n pho s pho- E RK s t a i n i ng ( as do c u me n te d by imm unoh ist o c h emistr y ) were do c u me n t ed i n t he ea rli e r tri al s bu t , i n retr o s p ect , t h e assa y s were no t se n siti ve enough t o d i s ti ngu i sh b etwee n m od est (~ 50 %) k i n ase i nh i b iti on v e r s u s m o r e co m p l e t e BRA F or MEK i nh i b iti on. Efficac y i n p recli n ical m od els is s i gn ifi can tl y im p r ov e d u si ng do ses t h at g i v e >80 % i nh i b iti on, and t h e hu m an tri a l da t a sugges t t h at t h is d e g ree o f p at h wa y b l o c k a d e is als o r e qu i r e d f o r a h i gh c li n i ca l r e s pon se rate . C o llecti v el y , t h ese e xp erie n c es ill u st r at e t he c riti ca l need f o r qu a n titati v e ph armac odyn amic assa y s t o meas ur e t a r ge t i nh i b iti on ea rly i n cli n ical d e v el op me n t . A sec ond less on is t h e im po rt ance o f geno t yp i ng all p atie n ts f o r m u tati on o r am p lificati on o f t h e r elev an t d r ug t a r ge t . No t o n l y do es t h is e n s u re t h at a s u f ficie n t nu m be r of p atie n t s w it h t he b i o m a r ke r o f i n terest are i n cl ud e d i n t h e st ud y , bu t a lso t h at t h e r esu lt s p r ov i de co m p elli ng e v i d e n ce earl y i n cli n ical d e v el op me nt i n s upp ort ( o r no t) o f t he p r ecl i n ical hypo t h esis .

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Getting It Right: ALK and Lung Cancer

nan nan

Getting It Right: ALK and Lung Cancer

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Getting It Right: ALK and Lung Cancer

nan nan

Th e d e ve l op m en t o f t he A L K i nh i b it o r criz o ti n i b (PF- 02341066 ) ill u stra tes how a n unexpec t ed s i gna l ob t a i n e d i n a small nu m b er o f p atie n ts ca n qu ic k l y sh ift a p r og r a m i n an en tirel y n ew d irecti on wit h a h i gh p r ob a b il ity of s u cce ss. T he key i ng r ed i en t is t h is st o r y is a familiar on e — a str ong m o lec u l a r hypo t hes i s backed up by cli n ical res pon se d ata i n a small numbe r of ca r e fu ll y se l ec t ed pa ti en t s. Criz o ti n i b eme r g e d fr o m a d r ug d isc ov er y progr am a t Pfi ze r t ha t was f o c u se d on fi nd i ng i nh i b it o rs o f t h e MET r ece p t or t y r os i ne k i nase and en tere d t h e cli n ic wit h t h is ta r g et as its lea d

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Getting It Right: ALK and Lung Cancer

nan nan

i nd icati on . As we p r ev i ous l y lear n e d wit h imati n i b, esse n tiall y all k i n a se i nh i b it ors have ac ti v it y aga i ns t o t h er tar g ets (s o calle d o ff-t a r g et acti v iti es ) , wh ic h c an so m e tim es p r ove t o b e a dv a n ta g e ou s . Off-ta r g et acti v ities are t yp icall y d i scove r ed by sc r een i ng c o m pound s a g ai n st a lar g e p a n el o f k i n ases to es t ab li sh p r o fil es o f relati v e selecti v it y a g ai n st t h e i n te nd e d ta r g et . O f f-t a r ge t ac ti v it y , po te n c y , a nd ph armace u tical p r op erties (b i o a v ail ab ilit y , ha lf-lif e ) a r e a ll fact o rs t h at i n fl u e n ce t h e d ecisi on o f w hich c o m pound t o advance t o c li n ical d e v el op me n t . T h e p rimar y o f f-ta r g et acti v it y o f c ri zo ti n i b i s aga i ns t t h e ALK t y r o si n e k i n ase . ALK was fir s t i den tifi ed as a ca nd i d ate d ri v er on c og e n e i n 1994 t h r o u gh t h e cl on i ng o f t he t( 2,5 ) ch r o m o s o mal tra n sl o cati on ass o ciate d wit h a n a p lasti c l a r ge ce ll l y m phom a , w h ic h creates t h e nu cle opho smi n /a n a p l astic l y m pho m a k i nase ( N P M - A L K ) f u si on g e n e T h is d isc ov er y , t og et h er with t h e d em ons tr a ti on t ha t N P M -ALK ca u ses l y m pho ma i n mice , ma d e a c o m p elli ng case f o r A L K as a d r ug ta r g et i n t h is d isease . B u t t h ere was limite d i n t e r es t i n deve l op i ng ALK i nh i b it o rs b eca u se t h is p artic u lar l y m pho m a sub t ype i s r a r e and m o st c o mm on l y f ound i n c h il d re n. ( C o m p a n i es a r e gene r a ll y r e l u cta n t t o d e v el op d r ug s s o lel y f o r p e d iatric i nd icati ons because o f co m p l ex ities relate d t o do se selecti on a nd a dd iti onal r e gu lat o r y gu i de li nes. E f f o rt s t o streamli n e t h is d e v el op me n t p r o cess are und e rwa y , such as t he C r ea ti ng H op e Act , w h ic h p r ov i d es n ew i n ce n ti ves for c o m pan i es t o pu r sue ped iatric i nd icati on s . ) I n 2007, a d iffere n t ALK fu si on gene ca ll ed E M L 4 - A LK was d isc ov ere d i n a small fracti on o f p atie n ts wit h l ung adenoca r c i no ma , wit h a n estimate d fre qu e n c y o f 1 % t o 5% Th i s d i scove r y d i d no t i m me d iatel y ca p t u re t h e atte n ti on o f d r ug d e v el op er s, bu t seve r a l academ ic g r oup s w ho h a d alrea dy b e gun testi ng l ung ca nce r pa ti en t s seen a t their i n stit u ti on s f o r EGFR m u tati on s sim p l y a dd e d a n E M L 4 - A L K f us i on t est t o t h e scree n i ng p a n el . Ast u te cli n ical i nv esti g at o r s pa rti c i pa ti ng i n th e ph ase I trial o f criz o ti n i b, w h ic h was d esi gn e d t o i nc l ude pa ti en t s w it h a b r o a d arra y o f a dv a n ce d ca n cers , we re a w a r e of t he o f f-t a r ge t A L K a cti v it y a nd e n r o lle d se v eral l ung ca n cer p atie n ts wit h E M L 4 - A L K f usion s i n t h e st ud y . T h ese p atie n ts h a d r ema rk a b l y d r a m a ti c r esponse s T h is sere nd i p it ou s fi nd i ng i n a few AL K -

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Extending the Model to RET Mutations in Thyroid Cancer: Clinical Responses, But Why?

nan nan

S ub sets o f pa ti en t s w it h pap illar y o r me du llar y t hy r o i d ca n cer h a v e acti v atin g m u t a ti ons o r tr ans l o cati on s tar g eti ng t h e RET t y r o si n e- k i n ase r ece p t o r , r a i s i ng t he ques ti on o f w h et h er RET i nh i b it o rs mi gh t h a v e a r ole i n t h is d i sease . A lt hough no d r ug s s p ecificall y d esi gn e d t o i nh i b it RET h a v e e n ter ed t he c li n i c, f ou r co m pound s wit h o f f-ta r g et acti v it y a g ai n st R ET (vande t an i b, so r a f en i b, m o tesa n i b, a nd ca bo za n ti n i b ) h a v e all s ho w n si ng le - ag en t ac ti v it y i n t hy r o i d ca n cer st ud ies – V a nd eta n i b a nd ca bo za n ti n i b a r e cu rr en tl y app r ov e d i n me du llar y t hy r o i d ca n cer b ase d on im prov e d p r og r ess i on -fr ee su r v i v al i n ph ase III re g istrati on trials .

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Extending the Model to RET Mutations in Thyroid Cancer: Clinical Responses, But Why?

nan nan

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Extending the Model to RET Mutations in Thyroid Cancer: Clinical Responses, But Why?

nan nan

Beca u se a ll f ou r co m pounds a ls o i nh i b it VEGF rece p t o r , it is un clear wh et h e r t he c li n i ca l bene fit ob ser v e d i n t h ese st ud ies is e xp lai n e d by i nh i b iti o n o f R E T , V E G F r ec e p t o r , o r bo t h. U n li k e t h e criz o ti n i b trials i n ALK-pos iti ve l ung cance r , enro llme n t i n t h ese re g istrati on st ud ies was n o t r est r icte d t o pa ti en t s w it h R E T m u tati on s . I n a dd iti on t o t h e fact t h at t hy r oid ca n ce r p ati en t s a r e no t r ou ti ne l y scree n e d f o r t h ese m u tati on s , t h e p rimar y r eas on f or i nc l ud i ng a ll co m er s i n t h ese st ud ies is t h at cli n ical res pon ses a r e ob se rv e d i n a l a r ge r fr ac ti on of p atie n ts t h a n ca n b e acc oun te d f o r b ase d on t h e s u s pec t ed fr equency o f an RET m u tati on. Res pon ses i n p atie n ts wit hout R ET m u t a ti on (if t hey occu r) mi gh t b e e xp lai n e d by m u tati on s i n o t h er g e n es i n t he RA S- MA P k i nase p at h wa y s u c h as BRAF o r HRAS , w h ic h a r e found i n a subs t an ti a l fr ac ti on o f p atie n ts a nd t yp icall y do no t ov erla p w ith R ET alter a ti ons . C l ea rl y , det aile d g e no t yp e/res pon se c o rrelati on s , as d em on st ra t ed i n l ung cance r and mela no ma , will clarif y t h e r o le o f t h ese

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Extending the Model to RET Mutations in Thyroid Cancer: Clinical Responses, But Why?

nan nan

m u tati ons i n p r ed i c ti ng t he r e s pon se t o t h ese d r ug s . T hy r o i d ca n cer is al so a c o m p elli ng i nd i ca ti on f o r t he B RAF a nd MEK i nh i b it o rs d isc u sse d pr e v i ous l y i n m e l ano m a.

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

F L T3 Inhibitors in Acute Myeloid Leukemia: Did the Genomics Mislead Us?

nan nan

F L T3 Inhibitors in Acute Myeloid Leukemia: Did the Genomics Mislead Us?

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

F L T3 Inhibitors in Acute Myeloid Leukemia: Did the Genomics Mislead Us?

nan nan

S hor tl y a ft e r t he success o f imati n i b, t h e rece p t o r t y r o si n e –k i n ase F L T 3 eme r g e d as a co m pe lli ng d r ug ca nd i d ate b ase d on t h e p rese n ce o f acti v a ting m u tati ons i n abou t one -t h ir d o f p atie n ts wit h ac u te m y el o i d le uk emia .

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

F L T3 Inhibitors in Acute Myeloid Leukemia: Did the Genomics Mislead Us?

nan nan

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

F L T3 Inhibitors in Acute Myeloid Leukemia: Did the Genomics Mislead Us?

nan nan

L a bor ator y s t ud i es docu m en te d t h at F L T 3 alleles b eari ng t h ese m u tati ons, wh ic h o c cu r as i n t e r na l t andem dup licati on s (ITD) o f t h e j ux tamem b ra ne do mai n o r a po i n t m u t a ti on i n t h e k i n ase do mai n, f un cti on as d ri v er on c og e n e s i n m ouse m ode l s, gi v i ng ph e no t yp es a n al ogou s t o BCR-AB L .

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

F L T3 Inhibitors in Acute Myeloid Leukemia: Did the Genomics Mislead Us?

nan nan

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

F L T3 Inhibitors in Acute Myeloid Leukemia: Did the Genomics Mislead Us?

nan nan

A s w it h R ET i n t hy r o i d canc e r , no c o m pound s h a d b ee n s p ecificall y op timiz ed t o t a r ge t F L T 3, bu t se v eral d r ug s wit h o f f-ta r g et F L T 3 acti v it y w e r e r e d ir ec t ed t o acu t e m ye l o i d le uk emia (AML) . Disa ppo i n ti ng l y , t h e f i r st t hree o f t he co m pounds t e ste d (mi do sta u ri n, lesta u rti n i b, a nd s un iti nib ) s how e d on l y m a r g i na l s i ng l e -a g e n t acti v it y i n rela p se d AML p atie n ts , e ven i n t ho se wit h F L T 3 m u t a ti ons. Des p ite t h e str ong m o lec u lar rati on ale f o r F L T3 as a d ri ve r l es i on, ques ti on s were raise d a bou t t h e v ia b ilit y o f F L T 3 as a drug t a r ge t . P ha rm acodyna mic st ud ies s ho we d e v i d e n ce o f F L T 3 k i n ase i nh i b iti o n i n t u m o r ce ll s, bu t t h e ma gn it ud e a nd du rati on o f t h ese e f fects w e r e d i f fi cu lt t o quan tif y , r a i s i ng t h e po ssi b ilit y o f i n a d e qu ate tar g et i nh i b iti o n I ndeed, t he dose o f all t h ree c o m pound s was limite d by t ox icitie s be li eved t o be i ndep e nd e n t o f F L T 3. A m o re p essimistic i n te rpr etati on was t ha t F L T 3, alt hough p res u ma b l y im po rta n t f o r t h e i n itiati on o f AM L , was no l ong er re qu ire d f o r t u m o r mai n te n a n ce du e t o the acc u m u l a ti on o f add iti ona l d r iv er g e no mic alterati on s . If tr u e , e v e n a c o m p let e F L T 3 b l ockade w it h a h i gh l y selecti v e i nh i b it o r w ou l d b e e xp ecte d t o f a il . Bu t t h i s v i ew was no t s uppo rte d by t h e fact t h at cli n ical r es pon s es we r e obse r ved i n t h e s o mew h at a n al ogou s sit u ati on o f si ng le-a g e n t A BL k i nase i nh i b it o r tr e atme n t o f CML i n b last crisis , w h ere BCR-

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

F L T3 Inhibitors in Acute Myeloid Leukemia: Did the Genomics Mislead Us?

nan nan

A B L is j us t one o f m any add iti on al g e no mic alterati on s t h at c on tri bu te t o d isease p r og r ess i on, ye t co m p lete remissi on s are ob ser v e d i n ma ny p atie nts. D es p it e t h i s pess imi s m abou t F L T 3 as a v ia b le d r ug ta r g et , se v eral d r ugs a r e now advanc i ng t owa r d d r ug re g istrati on trials . Mi do sta u ri n, on e o f t he ea r l y c ompounds t ha t showed d isa ppo i n ti ng si ng le-a g e n t acti v it y i n r ela p se d AM L , i s be i ng eva l ua te d i n a ra ndo mize d ph ase III trial i n n ew ly d ia gno se d AM L co m b i ned w it h sta nd ar d i ndu cti on c h em o t h era p y . A si ngle -a r m ph a se II s t udy showed h ig h er a nd m o re du ra b le remissi on rates i n F L T3 m u ta n t pa ti en t s when co m pa r ed t o h ist o rical c on tr o ls . T h e sec ond c o m pound, qu i za rti n i b ( AC220 ) , is a n e x t- g e n erati on F L T 3 i nh i b it o r with gr eate r po t ency and spec ifi c it y a nd wit h si ng le-a g e n t acti v it y i n F L T 3 m u ta n t re l apsed AM L —p r ec isel y t h e popu lati on w h ere mi do sta u ri n a nd o t h e r s fa il ed . T he f ac t t ha t s o me res pond er p atie n ts h a v e rela p se d wit h drug-r esi s t an t ga t ekeepe r m u tati on s i n t h e F L T 3 k i n ase do mai n p r ov i d es for mal p r oo f t ha t F L T 3 i s t he r ele v a n t ta r g et Ass u mi ng t h ese c o m pounds prov e s uccess f u l i n AM L , it will b e im po rta n t t o e x ami n e t h eir acti v it y i n t h e r a r e cases o f ped i a tri c acu te l y m pho i d le uk emia ass o ciate d wit h F L T 3 m u tati on. A lt hough t he j u r y i s still ou t on F L T 3 i nh i b it o rs , t h e fail u re o f ea r l y c ompounds i n AM L i s remi n isce n t o f t h e fail u res o f earl y RAF a nd M EK i nh i b it o r s i n m e l ano m a. C o llecti v el y , t h ese e x am p les em ph asize t he im por ta nce o f us i ng op timi zed c o m pound s t o test a m o lec u larl y b ase d hypo t h e s i s i n pa ti en t s and t o f o c u s e n r o llme n t on t ho se p atie n ts wit h t h e r ele v a n t m o l ecu l a r l es i on.

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Kidney Cancer: T argeting the T umor and the Host W ith Mammalian T arget of Rapamycin and VEGF Receptor Inhibitors

nan nan

Kidney Cancer: T argeting the T umor and the Host W ith Mammalian T arget of Rapamycin and VEGF Receptor Inhibitors

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Kidney Cancer: T argeting the T umor and the Host W ith Mammalian T arget of Rapamycin and VEGF Receptor Inhibitors

nan nan

A r ec urr i ng t he m e i n t h i s chap ter is t h e critical r o le o f d ri v er k i n ase m u tati ons i n gu i d i ng t he develop me n t o f k i n ase i nh i b it o rs . Ir on icall y , se v e r al k i nase i nh i b it o r s have b ee n a pp r ov e d f o r k i dn e y ca n cer ov er t h e p ast 5 y e a r s i n a t u m o r t ype wit h no kno w n k i n ase m u tati on s . T h e m o st c o mm on m o l ecu l a r a lt e r a ti on i n k i dn e y ca n cer is a l o ss o f f un cti on i n t he

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Kidney Cancer: T argeting the T umor and the Host W ith Mammalian T arget of Rapamycin and VEGF Receptor Inhibitors

nan nan

V on H i ppe l-Li ndau ( VH L) t u m o r s upp ress o r g e n e , res u lti ng i n t h e acti v atio n o f t he hypox i a i ndu ci b le fact o r p at h wa y As a c on se qu e n ce o f VHL l o s s, wh i ch no rm a ll y t a r g ets hypox ia-i ndu ci b le fact o r (HIF) p r o tei ns for pro t easo m a l deg r ada ti on, HIF- 1α a nd HIF- 2α are c on stit u ti v el y acti ve t r a n sc r i p ti on f ac t o r s t ha t f unct i on as on c og e n es t h r ough acti v ati on o f a n a rr a y of downs tr ea m t a r ge t g e n es . Am ong t h ese is t h e a ng i og e n esis fact o r VEG F , wh i ch i s sec r e t ed by HIF-e xp ressi ng cells a nd p r o m o tes t h e d e v el opmen t and m a i n t enance o f t u m o r n e ov asc u lat u re . HIF-me d iate d sec r eti on o f V E G F by t u m o r cells li k el y e xp lai n s t h e h i gh l y v asc u lar h ist op at ho l ogy o f c l ea r ce ll r e nal carci no ma . All t h ree c u rre n tl y a pp r ov e d a ng i og e nes i s i nh i b it o r s (t he mono cl on al a n ti body b e v aciz u ma b ta r g eti ng VEG F a nd t he k i nase i nh i b it o rs s o rafe n i b a nd s un iti n i b tar g eti ng o r its r ece p t or V E G F r ecep t o r) hav e si ng le-a g e n t cli n ical acti v it y i n clear cell ca r ci noma . T he h i gh speci ficit y o f b e v aciz u ma b f o r VEGF lea v es lit tle doub t t ha t t he ac ti v it y o f t h i s dr ug is e xp lai n e d by a n tia ng i og e n ic e f fects . I n c on t r ast , t he o f f-t a r ge t ac ti v iti e s o f s o rafe n i b a nd s un iti n i b i n cl ud e se v er al k i n ases e xp r essed i n k i dney t u m o r cells , str o ma , a nd i n flammat o r y cells (PDGFR, RA F , R E T , F L T 3, a n d o t h ers) . I n teresti ng l y , t h e p rimar y e f fec t o f b e v aciz u m ab i n k i dney cance r is d isease sta b ilizati on, w h ereas s o rafe n i b a nd s un i t i n i b have subs t an ti a l p artial res pon se rates . T h is raises t h e qu es tion of wh et he r t he supe ri o r an tit u m o r acti v it y o f t h e VEGF rece p t o r k i n ase i nh i b it ors i s due t o t he concu r r e n t i nh i b iti on o f o t h er k i n ases . H o we v e r , p a r tial r e sponses r a t es w it h n e x t- g e n erati on VEGF rece p t o r i nh i b it o rs ( a x iti n i b, pazopan i b, and ti voz a n i b ) , all o f w h ic h h a v e g reater po te n c y a nd selecti v it y f o r t he V E G F r ecep t o r , are similarl y h i gh, a nd rei n f o rce t h e im por ta nce o f t he V E G F r ecep t o r as t h e critical ta r g et i n k i dn e y ca n ce r

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Kidney Cancer: T argeting the T umor and the Host W ith Mammalian T arget of Rapamycin and VEGF Receptor Inhibitors

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EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Kidney Cancer: T argeting the T umor and the Host W ith Mammalian T arget of Rapamycin and VEGF Receptor Inhibitors

nan nan

Paz op a n i b i s app r oved f o r adv a n ce d k i dn e y ca n ce r , w h ereas a x iti n i b is a pprov e d as second -li ne t he r a p y . T wo i nh i b it o r s o f t he m a mmalia n ta r g et o f ra p am y ci n (m T OR) k i n ase ( temsi ro lim us and eve r o lim us) are als o a pp r ov e d f o r a dv a n ce d re n al cell ca r ci noma . Bo t h t e m s ir o li mu s a nd e v er o lim u s are kno w n as ra p al og s b eca u se bo t h a r e che mi ca l de ri v ati v es o f t h e n at u ral p r odu ct sir o lim u s (r a p am yc i n ) . Sir o lim us was a p p r ov e d m o re t h a n 10 y ears a go t o p re v e n t

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Kidney Cancer: T argeting the T umor and the Host W ith Mammalian T arget of Rapamycin and VEGF Receptor Inhibitors

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gr a f t r eje c ti on i n tr ansp l an t r ec i p ie n ts b ase d on its imm uno s upp ressi v e prop e r tie s aga i ns t T ce ll s. Sir o lim u s als o h as po te n t a n ti p r o liferati v e e f f ects a g ai n st vascu l a r endo t he li a l ce lls a nd, on t h at b asis , is i n c o r po rate d i n t o drug- el ut i ng ca r d i ac s t en t s t o p re v e n t c o r on ar y arter y reste no sis f o ll o wi ng a ng i op l as t y . Rapa l ogs d i f f e r fr o m all t h e o t h er k i n ase i nh i b it o rs d isc u s sed i n t h is c hap t e r i n t ha t t hey i nh i b it t h e k i n ase t h r ough a n all o steric mec h a n ism r a t he r t han by t a r g eti ng t h e m T OR k i n ase do mai n. Beca u se r a p al og s a l so i nh i b it t he g r owth o f ca n cer cell li n es fr o m d i f fere n t tiss u es o f or i g i n, c l i n i ca l tri a l s we r e i n it i ate d t o st udy t h eir po te n tial r o le as a n tica nce r a g e n ts i n a b r oad r ange o f t u m o r t yp es . Base d on res pon ses i n a few ph a se I p atie n ts wit h d i f f e r en t t u m o r t yp es (i n cl ud i ng k i dn e y ca n cer) , e xp l o rat o ry ph ase II s t ud i es we r e conduc t ed i n se v eral d iseases . Si ng le-a g e n t acti v it y o f temsi ro lim us was obse r ved i n a ph ase II k i dn e y ca n cer st ud y t h e n c onf i r me d i n a phase III r eg i s trati on trial . T h e ph ase III e v er o lim u s tria l, wh ic h was i n iti a t ed a ft e r t e msir o lim u s , was no tew o rt hy b eca u se cli n ical b e n e f it was de m ons tr a t ed i n patie n ts w ho h a d p r og resse d on t h e VEGF r ece p t or i nh i b it o r s so r a f en i b or s un iti n i b . In p ar a ll e l w it h t he e m p irical cli n ical d e v el op me n t o f ra p al og s , v ari ous la bor at o ri es exp l o r ed t he m o lec u lar b asis f o r mTOR d e p e nd e n ce i n ca n c e r cells . m T OR f unc ti ons a t t he ce n ter o f a c o m p le x n etw o r k t h at i n te g rate s si gn als fro m g r ow t h f ac t o r r e ce p t o rs a nd nu trie n t se n s o rs t o re gu late cell grow t h and s i ze ( . I t do es s o, i n p art , by c on tr o lli ng t h e tra n sla tion of v a r i ous m RNAs w it h co m p le x 5 ′ un tra n slate d re g i on s i n t o p r o tei n. m T O R e x i s t s i n t wo d i s ti nc t co m p le x es kno w n as T OR c o m p le x 1 ( T O RC 1 ) and T ORC2. Rapa lo g s on l y i nh i b it t h e T ORC 1 c o m p le x, w h i ch is la r g el y respons i b l e f o r downs tream pho s pho r y lati on o f ta r g ets s u c h as S 6K1 / 2 and 4 E B P 1 / 2 t ha t r egu late p r o tei n tra n slati on . T h e T ORC 2 c o m p le x con tri bu t es t o t he act i v ati on o f AKT by pho s pho r y lati ng t h e im por ta n t r egu l a t o r y se ri ne r e si du e S 473 a nd is un affecte d by ra p al og s .

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Kidney Cancer: T argeting the T umor and the Host W ith Mammalian T arget of Rapamycin and VEGF Receptor Inhibitors

nan nan

T wo hypo t heses have e m e r g e d t o e xp lai n t h e cli n ical acti v it y o f ra p al ogs i n k i dn e y cance r . T he an ti p r o liferati v e acti v it y o f t h ese c o m pound s a g ai nst e ndo t h e l i a l ce ll s sugges t s an a n tia ng i og e n ic mec h a n ism , w h ic h is c on sist ent w it h t h e c li n i ca l ac ti v it y o f t h e VEGF rece p t o r i nh i b it o rs . B u t ra p al og s a lso i nh i b it th e g r ow t h o f k i dney c a n cer cell li n es i n la bo rat o r y m od els w h er e t h e e f f e c t s on t u m o r ang i ogen esis h a v e b ee n elimi n ate d. I n teresti ng l y , mR NA s f o r H IF 1 / 2 a r e a m ong t ho se w ho se tra n slati on is im p aire d by r a p al og s , and t h i s e f f ec t has be e n im p licate d as t h e p rimar y mec h a n ism o f r a p al og ac ti v it y i n k i dney can cer x e nog raft m od els . As wit h t h e VEGF r ece p t or i nh i b it o r s, a de t a il ed m o lec u lar a nno tati on o f t u m o rs fr o m r es ponde r s and non r esponde r s will s h e d li gh t on t h ese iss u es .

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Other Indications for m T OR Inhibitors: B r east Cancer and T ube r ous Scle r osis Complex Mutant Cancers

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Other Indications for m T OR Inhibitors: B r east Cancer and T ube r ous Scle r osis Complex Mutant Cancers

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Other Indications for m T OR Inhibitors: B r east Cancer and T ube r ous Scle r osis Complex Mutant Cancers

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T wo o t he r i nd i ca ti ons f o r m T OR h a v e eme r g e d, bo t h b ase d on f und ame ntal i n si gh ts f r o m l abo r a t o r y s t ud ies bu t fr o m qu ite d iffere n t a ng les . Precli n ic al st ud ies o f es tr ogen r ecep t o r (ER) t h era py i n b reast ca n cer s ugg este d t h at pho s ph ati dy li nos it o l 3 - k i nase (PI 3 K) p at h wa y acti v ati on ma y b e a mec h a n ism o f r es i s t ance and t h at t h is resista n ce c ou l d b e p re v e n te d o r ov e r c o m e by co m b i ned tr ea t m e n t wit h ER- b ase d d r ug s a nd ra p al og s s u c h as e v e ro lim us. Based on ev i den ce t h at s o me w o me n wit h p r og ressi v e d isease w h il e r ece i v i ng t he a romatase i nh i b it o r letr o z o le h a v e cli n ical b e n e f it f r o m t he add iti on o f ev er o lim u s , ra ndo mize d trials were i n itiate d c o m p a r i ng eve r o lim us + exe mesta n e t o e x emesta n e al on e (calle d B OLE R O - 2 ) , o r eve r o lim us + tam ox ife n t o tam ox ife n al on e (calle d T A MR AD ) . Bo t h s t ud i es de m on strate d s ub sta n tial im p r ov eme n ts i n time to progr ess ion i n wo m en w it h metastatic b reast ca n cer w ho h a d alrea dy fai led on e a ro m a t ase i nh i b it o r , res u lti ng i n FDA a pp r ov al o f t h e e v e ro lim us / exe m es t ane co m b i n ati on. E v i d e n ce o f cr o ss-tal k b etwee n t he P I3K p at hway and ho rm one rece p t o r si gn ali ng (ER i n b reast ca n ce r , a ndrog e n r ecep t o r i n p r os t a t e ca n cer) p r ov i d es a m o lec u lar rati on ale f o r the

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Other Indications for m T OR Inhibitors: B r east Cancer and T ube r ous Scle r osis Complex Mutant Cancers

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EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Other Indications for m T OR Inhibitors: B r east Cancer and T ube r ous Scle r osis Complex Mutant Cancers

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i nh i b it or o f an ups tr ea m k i nas e i n t h e fee db ac k l oop, s u c h as HER k i n as es

EAR L Y SUCCESS E S: T ARGET I NG CANCERS WITH W ELL - K NOWN K INASE M U T A TIONS (BCR-ABL, KI T , HER2)

Other Indications for m T OR Inhibitors: B r east Cancer and T ube r ous Scle r osis Complex Mutant Cancers

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or t h e i n s u li n li ke g r ow t h f ac t o r rece p t o r (IGFR) , t o b l o c k t h is und esire d e f f ect of r apa l ogs on PI 3K ac ti v ati on

DIRECT L Y T ARGETING T H E P I3K P A T H W A Y

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DIRECT L Y T ARGETING T H E P I3K P A T H W A Y

DIRECT L Y T ARGETING T H E P I3K P A T H W A Y

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M u tati ons o r copy nu m be r a lterati on s (e .g., am p lificati on o r d eleti on o f on c og e n e s o r t u m o r supp r esso r g e n es) i n PI 3 K p at h wa y g e n es (PIK 3 CA, P IK3 R 1, PTE N, AK T 1, and o t h ers) are am ong t h e m o st c o mm on a bnor m a liti es i n cance r . Cons e qu e n tl y , i n te n si v e e f f o rts at ma ny ph a r ma ceu ti ca l co m pan i es hav e b ee n d e vo te d t o t h e d isc ov er y o f small-m o lec u l e i nh i b it o r s t a r ge ti ng k i n ases i n t h e PI 3 K p at h wa y . I nh i b it o rs o f P I3K, A K T , and A TP- co m pe titi v e (rat h er t h a n all o steric) i nh i b it o rs o f m T O R t ha t t a r ge t bo t h t he T ORC 1 a nd T ORC 2 c o m p le x are all i n cli n ic al d e v el opmen t . P hase I c li n i ca l trials h a v e , i n g e n eral , esta b lis h e d t h at t h e p at hw a y can be e f fi c i en tl y t a r g ete d wit hou t seri ou s t ox icit y o t h er t h a n easil y ma nageab l e e f f ec t s on g l u c o se meta bo lism (w h ic h is a n tici p ate d b ase d on t he im po rt ance o f PI 3 K si gn ali ng i n i n s u li n si gn ali ng ) . Unfor t una t e l y , t he r e has been no e v i d e n ce t o d ate o f d ramatic si ng le-a g e nt cli n ical a c ti v it y w it h any o f t h ese a g e n ts , alt hough earl y res u lts wit h PI 3K al ph a sel ec ti ve i nh i b it o r BY L7 19 i n PIK 3 CA m u ta n t b reast ca n cer a pp e a r pro misi ng. Howeve r , t he fir s t app r ova l o f a d irect PI 3 K i nh i b it o r i n ca n cer is li k e ly t o c o me i n ch r on i c l y m phocy tic le uk emia a nd i n l y m pho ma , bu t no t on t he b asis of t u m o r geno mi cs. No rmal a nd mali gn a n t B cells are d e p e nd e n t on P I3K d elt a as we ll as B r u t on t y r o si n e k i n ase (BTK) f o r p r o liferati on a nd s urv i v al , r a i s i ng t he poss i b ilit y t h at i nh i b it o rs o f t h ese k i n ases mi gh t b e bro a d l y ac ti ve i n B - ce ll m a li g na n cies . C on cer n s a bou t t ox icit y on no rma l B cells w e re a ll ev i a t ed, i n pa rt , by t h e earlier cli n ical s u ccess o f t h e CD 20 a n ti body rit ux im ab i n l y m pho ma , w h ic h als o elimi n ates no rmal circ u lati ng B cells , bu t w it hou t s i gn ifi can t cli n ical se qu elae . T h e first s u c h PI 3 K d el ta i nh i b it o r , i de l a li s i b, has shown im p ressi v e acti v it y i n i ndo le n t non -H odg k in l y m pho m a as a s i ng l e agen t and i n rela p se d c h r on ic l y m pho c y tic le uk e mia

DIRECT L Y T ARGETING T H E P I3K P A T H W A Y

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wh e n g i ven i n co m b i na ti on w it h rit ux ima b. T h e BTK i nh i b it o r i b r u ti n i b, fo ll ow i ng a s imil a r c li n i ca l dev el op me n t p at h, was rece n tl y a pp r ov e d as sec ond- l ine t he r apy f o r ch r on ic l y m pho c y tic le uk emia a nd f o r ma n tle ce ll l y m pho m a

COMBIN A TI O NS O F K INASE IN H IB I T ORS T O INDUCT R E SP O NSE AND PREV E NT RES I S T ANCE

nan nan

COMBIN A TI O NS O F K INASE IN H IB I T ORS T O INDUCT R E SP O NSE AND PREV E NT RES I S T ANCE

COMBIN A TI O NS O F K INASE IN H IB I T ORS T O INDUCT R E SP O NSE AND PREV E NT RES I S T ANCE

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P r ecli n ic a l s t ud i es i nd i ca t e t ha t c o m b i n ati on s o f k i n ase i nh i b it o rs are r e qu i r e d t o r ea li ze t he ir f u ll po te n tial as a n tica n cer a g e n ts . T h e m o st c o mm on r a ti ona l e i s t o add r e ss t h e p r ob lem o f c on c u rre n t m u tati on s i n d i f f e r e n t pa t hways t ha t a ll ev iate d e p e nd e n ce on a si ng le- d ri v er on c og e ne. Th e b es t exa m p l es a r e cance rs wit h m u tati on s i n bo t h t h e RAS/MAP k i nase p at hw a y ( RA S o r BRA F) and t h e PI 3 K p at h wa y (PIK 3 CA o r PTEN) . I n m ou se mode l s, such doub l y m u ta n t t u m o rs fail t o res pond t o si ng le-a g e nt t r eatme n t w it h e it he r an AK T i nh i b it o r o r a MEK i nh i b it o r . H o we v e r , c o m b i na ti on tr ea tm en t can g ive d ramatic re g ressi on s . Similarl y , g e n eticall y eng i nee r ed mi ce t h at d e v el op KRAS- d ri v e n l ung ca n cer res pond on l y t o co m b i na ti on t he r apy w it h a PI 3 K i nh i b it o r a nd a MEK i nh i b it o r

COMBIN A TI O NS O F K INASE IN H IB I T ORS T O INDUCT R E SP O NSE AND PREV E NT RES I S T ANCE

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COMBIN A TI O NS O F K INASE IN H IB I T ORS T O INDUCT R E SP O NSE AND PREV E NT RES I S T ANCE

nan nan

T o d ate , c li n i ca l tri a l s co m b i ning d i f fere n t PI 3 K p at h wa y a nd RAS/MA P k i n ase pa t hway i nh i b it o r s hav e b ee n c h alle ng i ng du e t o t ox icities ass o cia ted w it h c on ti nuous, concu rr en t PI 3 K a nd RAS/MAP k i n ase p at h wa y i nh i b iti o n . Ma ny o f t he t u m o r t ypes di sc u sse d i n t h is c h a p ter do res pond t o t r eatme n t w it h a s i ng l e - agen t k i n ase , bu t rela p se d es p ite c on ti nu e d i nh i b i to r t h e r a p y . Resea r ch i n t o t he cau ses o f “ac qu ire d ” k i n ase i nh i b it o r resista nce h as r e v e a l ed t wo p rim a r y m ech a n isms: ( 1 ) nov el m u tati on s i n t h e k i n ase do mai n o f t he d r ug t a r ge t t ha t p recl ud e i nh i b iti on, o r ( 2 ) b y pa ss o f t h e dr i v e r kinase s i gna l by ac ti va ti on o f a p arallel k i n ase p at h wa y . I n bo t h cases , t h e so l u ti on i s co m b i na ti on t h era py t o p re v e n t t h e eme r g e n ce o f r esista nce. An e l egan t de m on strati on o f t h is a pp r o ac h c o mes fr o m CML

COMBIN A TI O NS O F K INASE IN H IB I T ORS T O INDUCT R E SP O NSE AND PREV E NT RES I S T ANCE

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wh e r e res i s t ance t o im a ti n i b is p rimaril y ca u se d by m u tati on s i n t h e BC R - A B L k i nase do m a i n . T he s ec ond - g e n erati on ABL i nh i b it o rs d asati n i b a nd n il o ti n i b a r e e f f ec ti ve aga i n st m o st imati n i b -resista n t BCR-ABL m u ta n ts and we r e i n iti a ll y app r ov e d as si ng le-a g e n t t h era py f o r imati n i b - r esista n t CM L . V e r y r ecen tl y , bo t h d r ug s h a v e p r ov e n s up eri o r t o imati n i b i n t he up fr on t tr ea tme n t o f CML du e t o i n crease d po te n c y a nd f e w e r m echan i s m s o f acqu ir ed resista n ce H o we v e r , on e BCR-AB L m u tati on ca ll ed T 315 I i s r es i st a n t t o all t h ree d r ug s . T h e t h ir d - g e n erati on A B L k i nase i nh i b it o r pona ti n i b b l o c k s T 315 I a nd s ho we d acti v it y i n a ph ase II c li n i ca l tri a l t ha t i nc l ud e d CML p atie n ts wit h t h e T 315 I m u tati on , r esu lti ng i n F DA a pp r ov al . H o we v e r , s ub se qu e n t re po rts o f se v e r e vascu l a r occ l us i ve even ts , s u c h as str ok e a nd h eart fail u re , le d t o w it hdr a wa l fr o m t he m a r ke t , fo ll o we d by a pp r ov al f o r restricte d u se i n T315I- m u t an t pa ti en t s. Ana l o g ou s a pp r o ac h es are ongo i ng i n o t h er d ise ases s u c h as EG F R -m u t an t l ung c a n ce r , w h ere ac qu ire d resista n ce t o t h e fron tli ne k i nase i nh i b it o r i s a ls o ass o ciate d wit h m u tati on s i n t h e ta r g et k i n ase . Pr o mi s i ng c li n i ca l res u lts h a v e b ee n re po rte d wit h irre v ersi ble EG FR i nh i b it o r s such as CO - 1686 a nd AZD 9291. Th e c li n i ca l deve l op m en t o f k i n ase i nh i b it o r c o m b i n ati on s t o p re v e n t ac qu i r ed r es i s t ance i s r e l a ti ve l y strai gh tf o rwar d. Beca u se t h e fr on tli n e d r ug is al r ea dy app r oved, success wou l d b e d etermi n e d by a n im p r ov eme n t in r es pon s e du r a ti on us i ng t he co m b i n ati on. T h e sit u ati on is m o re c o m p le x wh e n t wo expe rim en t a l co m pound s (e .g., a PI 3 K p at h wa y i nh i b it o r a nd a M EK i nh i b it o r) a r e co m b i ned, n eit h er o f w h ic h s ho ws si gn ifica n t si ng le - a g e n t acti v it y . O l de r r egu l a t o r y gu i d eli n es re qu ire d a f ou r -arm st udy t h a t c o m p a red each s i ng l e agen t t o t h e c o m b i n ati on a nd t o a c on tr o l g r oup i n ord e r t o ob t a i n app r ova l o f t h e c o m b i n ati on. Rec ogn izi ng t h at t h is d esi gn c ou l d d i scou r age d r ug deve l op ers as well as p atie n ts fr o m m ov i ng f o rw a r d b eca u se it r equ ir es a l a r ge sa m p le size , t h e FDA h as iss u e d n ew gu i d eli nes for t h e d e ve l op m en t o f nove l co m b i n ati on s t h at re qu ire a tw o -arm r e g ist r ati on s t udy co m pa ri ng th e c o m b i n ati on t o sta nd ar d o f care

COMBIN A TI O NS O F K INASE IN H IB I T ORS T O INDUCT R E SP O NSE AND PREV E NT RES I S T ANCE

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COMBIN A TI O NS O F K INASE IN H IB I T ORS T O INDUCT R E SP O NSE AND PREV E NT RES I S T ANCE

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op timiz a ti on and dose schedu le t h at is n ee d e d t o safel y c o m b i n e tw o i nv esti g ati ona l d r ugs. Much li k e t h e d e v el op me n t o f c o m b i n ati on c h em o t he r apy seve r a l decade s a go, it ma y b e im po rta n t t o select c o m pounds w it h nonove rl app i ng t ox icities t o all o w f o r s u fficie n t do ses o f eac h dr u g t o be ach i eved.

SPECUL A TIONS O N THE FU T URE ROLE OF KINASE IN H IBI T ORS IN CANCER MEDICINE

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SPECUL A TIONS O N THE FU T URE ROLE OF KINASE IN H IBI T ORS IN CANCER MEDICINE

SPECUL A TIONS O N THE FU T URE ROLE OF KINASE IN H IBI T ORS IN CANCER MEDICINE

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Th e ro le o f geno mi cs i n p r ed i c ti ng a res pon se t o k i n ase i nh i b it o r t h era py is now i rr e fu t ab l e. As t he nu m b er o f k i n ase d ri v er m u tati on s c on ti nu es t o gro w , t he fi e l d i s li ke l y t o m ov e awa y fr o m t h e c u rre n t strate gy o f a c ompan i on d i agnos ti c f o r eac h d r ug. Rat h e r , c o m p re h e n si v e m u tati on al prof ili ng p l a tf o rm s t ha t que r y eac h t u m o r f o r hund re d s o f po te n tial ca n c e r m u tati ons a r e m o r e li ke l y t o eme r g e as t h e d ia gno stic p latf o rm . T h e numbe r of d i r ect ly ac ti onab l e m u t a ti on s (mea n i ng t h e p rese n ce o f a m u tati on d e f i n es a tr ea tm en t dec i s i on suppo rte d by cli n ical trial d ata) remai n s l o w , bu t t h is nu m be r w ill undoub te d l y g r o w . I n a dd iti on, it is b ec o mi ng a pp a rent t h at ma ny pa ti en t s have r a r e m u tati on s ( d efi n e d as rare i n t h at h ist o l og ic t u m or ty pe ) bu t a r e, i n t heo r y , acti on a b le . Beca u se t h ese e x am p les are un li k el y t o be f o rm a ll y eva l u ate d i n cli n ical trials , ma ny ce n ters h a v e op e n e d baske t s t ud i es ( w it h el i g i b ilit y b ase d s o lel y on m u tati on p r o file) t o ca p t ur e these cases w it h so me re po rts o f remar k a b le s u ccess . M ore e f f o rt m us t be devo t ed t o ma n i pu lati ng t h e do se a nd sc h e du le o f k i n ase i nh i b it o r t he r apy t o ma x imize efficac y a nd mi n imize t ox icit y . T o d ate , all k i nase i nh i b it o r s hav e b ee n d e v el op e d b ase d on t h e ass u m p ti on that a 24 / 7 c ove r age o f t he t a r ge t is re qu ire d f o r e f ficac y . C on se qu e n tl y , m o s t c o m pounds a r e op timi zed t o h a v e a l ong ser u m h alf-life ( 12 t o 24 hou rs) . P h ase II doses a r e t hen se l ec te d b ase d on t h e ma x im u m t o lerate d do se d ete r mi ned w it h da il y ad mi n istrati on. B u t a rece n t cli n ical o f t h e ABL i nh i b it or dasa ti n i b i n CM L i nd icates t h at e qu i v ale n t a n tit u m o r acti v it y c an b e ac h ie ved w it h i n t e rmitt en t th era p y B y g i v i ng la r g er do ses

SPECUL A TIONS O N THE FU T URE ROLE OF KINASE IN H IBI T ORS IN CANCER MEDICINE

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i n te r mit ten tl y , h i ghe r peak d r ug c on ce n trati on s were ac h ie v e d t h at res u lt ed i n e qu i va l en t and poss i b l y sup eri o r e f ficac y Similar res u lts were ob se rv e d i n l abo r a t o r y s t ud i e s o f EGFR i nh i b it o rs i n EGFR-m u ta n t l ung ca n ce r . C li n i ca ll y r obus t , quan titati v e assa y s o f tar g et i nh i b iti on are n ee ded t o h aste n p r og r ess i n t h i s a r ea. A lt hough t he f ocus o f t h i s c h a p ter is k i n ase i nh i b it o rs , t h e t h emes d e v el op e d he r e shou l d app l y br o a d l y t o i nh i b it o rs o f o t h er ca n cer tar g et s. Inh i b it ors o f t he G - p r o t e i n coup le d rece p t o r sm oo t h e n e d (SMO) i n p atie nts w it h met as t a ti c basa l ce ll ca rci no ma o r me du ll ob last o ma esta b lis h t h at t he dr i v e r mu t a ti on hypo t hes i s ext e nd s b e yond k i n ase i nh i b it o rs . SMO is a c o m ponen t i n t he Hedgehog pat h wa y , w h ic h is c on stit u ti v el y acti v ate d in s ub sets of pa ti en t s w it h basa l c ell carci no ma a nd me du ll ob last o ma du e t o m u tati ons i n t he Hedgehog liga nd - b i nd i ng rece p t o r Patc h e d - 1. T reatme nt w it h t h e S MO i nh i b it o r v i s m od e g i b le d t o im p ressi v e res pon ses i n b asal cell ca r ci noma and m edu ll ob l as t om a p atie n ts w ho se t u m o rs h a d Patc h e d - 1 m u tati ons , , r esu lti ng i n F D A a pp r ov al . Ot h er nov el ca n cer tar g ets ar e eme r g i ng fr o m cance r geno me se qu e n ci ng p r o jects . S o matic m u tati on s i n t h e Kr e bs cyc l e enzy m e i soc itrate d e hyd r og e n ase (IDH 1 / 2 ) were f ound i n s ub sets of pa ti en t s w it h g li ob last o ma , AML , c hond r o sarc o ma , a nd c ho la ngioca r c i no m a , and t h e first IDH 2 i nh i b it o r h as e n tere d cli n ic al t r ials i n l euke mi a. Mu t a ti ons i n e n z y mes i nvo l v e d i n c h r o mati n rem od el ing, s u c h as the h i s t one m e t hy ltr an sferase EZH 2, h a v e b ee n re po rte d i n l y m pho m a and have spu rr ed the ongo i ng d e v el op me n t o f EZH 2 i nh i b it ors I nh i b it o r s o f ano t h er h ist on e met hy ltra n sferase DOT 1 L , wh ic h is r equ ir ed f o r t he m a i n te n a n ce o f mi x e d li n ea g e le uk emia (MLL) fu si on l euke mi as, a r e a l so i n cli n ical d e v el op me n t . Ki n ase i nh i b it o r s a r e j u st the fir s t wave o f m o l e c u larl y tar g ete d d r ug s u s h ere d i n by ou r und e r sta nd i ng o f t he m o l ecu lar und er p i nn i ng s o f ca n cer cells . T h ere is m u c h m o r e t o f o ll o w .

SPECUL A TIONS O N THE FU T URE ROLE OF KINASE IN H IBI T ORS IN CANCER MEDICINE

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Sawyers CL. Shifting paradigms: the seeds of oncogene addiction. Nat Med 2009;15(10): 1 158– 1 161.

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SPECUL A TIONS O N THE FU T URE ROLE OF KINASE IN H IBI T ORS IN CANCER MEDICINE

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Motzer RJ, Hutson TE, T omczak P , et al. Sunitinib versus interferon alfa in metastatic renal-cell carcinoma. N Engl J Med 2007;356(2): 1 15–124. Rini BI, W ilding G, Hudes G, et al. Phase II study of axitinib in sorafenib-refractory metastatic renal cell carcinoma. J Clin Oncol 2009;27(27):4462–4468. Sonpavde G, Hutson TE, Sternbe r g CN. Pazopanib, a potent orally administered small-molecule multita r geted tyrosine kinase inhibitor for renal cell carcinoma. Expert Opin Investig Drugs 2008;17(2):253–261. Bha r gava P , Esteves B, Al-Adhami M, et al. Activity of tivozanib ( A V -951) in patients with renal cell carcinoma (RCC): Subgroup analysis from a phase II randomized discontinuation trial (RDT). J Clin Oncol 2010;28(suppl):15s. Hudes G, Carducci M, T omczak P , et al. T emsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med 2007;356(22):2271–2281. Motzer RJ, Escudier B, Oudard S, et al. Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebo-controlled phase III trial. Lancet 2008;372(9637):449–456. McKeage K, Murdoch D, Goa FL. The sirolimus-eluting stent: a review of its use in the treatment of coronary artery disease. Am J Ca r diovasc Drugs 2003;3(3):2 1 1–230. Atkins MB, Hidalgo M, Stadler WM, et al. Randomized phase II study of multiple dose levels of CCI-779, a novel mammalian ta r get of rapamycin kinase inhibito r , in patients with advanced refractory renal cell carcinoma. J Clin Oncol 2004;22(5):909–918. Guertin DA, Sabatini DM. Defining the role of mTOR in cance r . Cancer Cell 2007;12(1):9–22. Thomas G V , T ran C, Mellingho f f IK, et al. Hypoxia-inducible factor determines sensitivity to inhibitors of m T OR in kidney cance r . Nat Med 2006;12(1):122–127. Bachelot T , Bou r gier C, Cropet C, et al. Randomized phase II trial of everolimus in combination with tamoxifen in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer with prior exposure to aromatase inhibitors: a GINECO stud y . J Clin Oncol 2012;30(22):2718–2724. Baselga J, Campone M, Piccart M, et al. Everolimus in postmenopausal hormone-recepto r -positive advanced breast cance r . N Engl J Med 2012;366(6):520–529. Carver BS, Chapinski C, W ongvipat J, et al. Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cance r . Cancer Cell 20 1 1;19(5):575–586. Krueger DA, Care MM, Holland K, et al. Everolimus for subependymal giant-cell astrocytomas in tuberous sclerosis. N Engl J Med 2010;363(19):1801–18 1 1. Iyer G, Hanrahan AL, Milowsky MI, et al. Genome sequencing identifies a basis for everolimus sensitivit y . Science 2012;338(6104):221. O’Reilly KE, Rojo F , She QB, et al. m T OR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 2006;66(3):1500–1508. Gonzalez-Angulo AM, Juric D, A r gilis G, et al. Safet y , pharmacokinetics, and preliminary activity of the alpha-specific P13K inhibitor BYL719: results from the first-in-human stud y . J Clin Oncol 2013;31(15 Suppl):2531. Byrd JC, Furman RR, Coutre SE, et al. T a r geting BTK with ibrutinib in relapsed chronic lymphocytic leukemia. N Engl J Med 2013;369(1):32–42. W ang ML, Rule S, Martin P , et al. T a r geting BTK with ibrutinib in relapsed or refractory mantle-cell lymphoma. N Engl J Med 2013;369(6):507–516. She QB, Halilovic E, Y e Q, et al. 4E-BP1 is a key effector of the oncogenic activation of the AKT and ERK signaling pathways that integrates their function in tumors. Cancer Cell 18(1):39–51.

SPECUL A TIONS O N THE FU T URE ROLE OF KINASE IN H IBI T ORS IN CANCER MEDICINE

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Engelman JA, Chen L, T an X, et al. E f fective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA H1047R murine lung cancers. Nat Med 2008;14(12):1351–1356. Gorre ME, Mohammed M, Ellwood K, et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 2001;293(5531):876–880. Shah N P , Nicoll JM, Nagar B, et al. Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell 2002;2(2): 1 17–125. Shah N P , T ran C, Lee F Y , et al. Overriding imatinib resistance with a novel ABL kinase inhibito r . Science 2004;305(5682):399–401. T alpaz M, Shah N P , Kantarjian H, et al. Dasatinib in imatinib-resistant Philadelphia chromosome-positive leukemias. N Engl J Med 2006;354(24):2531–2541. Kantarjian H, Shah N P , Hochhaus A, et al. Dasatinib versus imatinib in newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2010;362(24):2260–2270. Sawyers CL. Even better kinase inhibitors for chronic myeloid leukemia. N Engl J Med 2010;362(24):2314–2315. Saglio G, Kim D W , Issaragrisil S, et al. Nilotinib versus imatinib for newly diagnosed chronic myeloid leukemia. N Engl J Med 362(24):2251–2259. Cortes JE, Kim D W , Pinilla-Ibarz J, et al. A phase 2 trial of ponatinib in Philadelphia chromosome-positive leukemias. N Engl J Med 2013;369(19):1783–1796. Pao W , Miller V A, Politi KA, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2005;2(3):e73. Antonescu CR, Besmer P , Guo T , et al. Acquired resistance to imatinib in gastrointestinal stromal tumor occurs through secondary gene mutation. Clin Cancer Res 2005; 1 1( 1 1):4182–4190. Shah N P , Kantarjian HM, Kim D W , et al. Intermittent ta r get inhibition with dasatinib 100 mg once daily preserves e f ficacy and improves tolerability in imatinib-resistant and -intolerant chronic-phase chronic myeloid leukemia. J Clin Oncol 2008;26(19):3204–3212. Shah N P , Kasap C, W eier C, et al. T ransient potent BCR-ABL inhibition is sufficient to commit chronic myeloid leukemia cells irreversibly to apoptosis. Cancer Cell 2008;14(6):485–493. V on Ho f f DD, LoRusso PM, Rudin CM, et al. Inhibition of the hedgehog pathway in advanced basal-cell carcinoma. N Engl J Med 2009;361(12): 1 164– 1 172. Rudin CM, Hann CL, Laterra J, et al. T reatment of medulloblastoma with hedgehog pathway inhibitor GDC-0449. N Engl J Med 2009;361(12): 1 173– 1 178. Parsons D W , Jones S, Zhang X, et al. An integrated genomic analysis of human glioblastoma multiforme. Science 2008;321(5897):1807–1812. Mardis ER, Ding L, Dooling DJ, et al. Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med 2009;361( 1 1):1058–1066. W ard PS, Patel J, W ise DR, et al. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer Cell 2010;17(3):225–234. McCabe M T , Ott HM, Ganji G, et al. EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Natu r e 2012;492(7427):108– 1 12. Morin RD, Johnson NA, Severson TM, et al. Somatic mutations altering EZH2 ( T yr641) in follicular and di f fuse la r ge B-cell lymphomas of germinal-center origin. Nat Genet 2010;42(2):181–185. Bernt KM, Zhu N, Sinha AU, et al. MLL-rearranged leukemia is dependent on aberrant H3K79 methylation by DOT1L. Cancer Cell 20 1 1;20(1):66–78.

SPECUL A TIONS O N THE FU T URE ROLE OF KINASE IN H IBI T ORS IN CANCER MEDICINE

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Daigle SR, Olhava EJ, Therkelsen CA, et al. Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibito r . Cancer Cell 20 1 1;20(1):53–65.

SPECUL A TIONS O N THE FU T URE ROLE OF KINASE IN H IBI T ORS IN CANCER MEDICINE

Histone De a ce t y l ase Inh i b i tors and De me thy l ating Ag e n t s

nan nan

Histone De a ce t y l ase Inh i b i tors and De me thy l ating Ag e n t s

IN T RODUCTION

nan nan

IN T RODUCTION

nan nan

How e v e r , t a r ge ti ng ep i gene tic p r o cesses t o do w n re gu late t h e acti on o f ov e r e xp r essed genes i s a l so an eme r g i ng area o f researc h . T h is c h a p ter d esc r i b e s t he bas i s o f ep i gene tic c h a ng es i n ca n cer a nd d isc u sses s o me o f t h e latest app r oaches t ha t t a r g et e p i g e n etic a bno rmalities i n ca n ce r

IN T RODUCTION

nan nan

IN T RODUCTION

nan nan

i n cl ud i ng t hose des i gned t o i ndu ce t h e ree xp ressi on o f sile n ce d g e n es , f o r ca n ce r t he r ap y . T he t wo app roac h es m o st mat u re i n d e v el op me n t are t h e

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

nan nan

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

nan nan

r e v e r se d. T hus, t he r apeu ti c r ep r og rammi ng o f p atter n s o f g e n e e xp ressi on c ou l d t heo r e ti ca ll y r esu lt i n a l ong -term c h a ng e i n t h e ca n cer cell ph e no t ype, even a ft e r t he i ndu ci ng d r ug s are rem ov e d, alt hough t o d ate , this h as no t been acco m p li shed. Th e funda m en t a l un it t ha t determi n es e p i g e n etic states is t h e nu cle o s ome t h at c on t a i ns an oc t a m e r o f h ist on e p r o tei n s ar ound w h ic h a pp r ox imatel y 160 b as e pa ir s o f DNA a r e w ra pp e d It is t h e po siti on i ng o f t h ese st ru ct ures, and t he t h r ee - d im en si on al as p ects o f t h eir s p aci ng, a nd t h e r e gu lati on o f t h i s p r ocess by po sttra n slati on al m od ificati on s o f t h e c on stit u e n t h i s t ones t ha t unde r p i n s t h e f un cti on s o f t h e e p i g e no me .

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Abnormal Gene Silencing

nan nan

On e k e y a lt e r a ti on i n cance r , w h ic h ca n b e ass o ciate d wit h altere d e p i g e n et ic con tr o l , i s abno rmal g e n e sile n ci ng. N o rmall y , s u c h sile n ci ng is fund am en t a l and r equ ir ed a t t h e le v el o f c h r o mati n a nd DNA met hy lati on r e gu lati on f o r t he lif e o f m u lt i cell u lar e uk ar yo tic o r g a n isms . T h e sile n ci ng is c r itical f o r r egu l a ti ng im por ta n t b i o l og ic p r o cesses , i n cl ud i ng all as p e cts of d e v el op m en t , d i f f e r en ti a ti on, im p ri n ti ng, a nd sile n ci ng o f lar g e c hro m oso m a l do m a i ns, i nc l ud i ng t h e X c h r o m o s o me o f female mammals .

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Abnormal Gene Silencing

nan nan

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Abnormal Gene Silencing

nan nan

F or e x am p l e, t he d i ve r s it y o f str u ct u re a nd f un cti on o f cells d eri v e d fr om e p it h eli a l o r m esenchy m a l o ri g i n, u ltimatel y d i f fere n tiati ng i n t o cells li n i ng t h e i n tes t i ne o r l ung o r f o rmi ng mat u re g ra nu l o c y tes a nd m yo c y tes , res ult fro m h e r it ab l e changes i n gene e xp ressi on t h at are no t t h e res u lt o f a c h a nge i n DNA sequence. A lt hough in ma ny s p ecies , sile n ci ng ca n b e i n itiate d and mai n tai ned so l e l y by p r ocess es i nvo l v i ng t h e c ov ale n t m od ificati on s o f h ist on es and o t he r ch r o m a ti n co m pon e n ts , v erte b rates u tilize a n a dd iti onal la y e r of gene r egu l a ti on. T h i s p r o cess i nvo l v es t h e on l y n at u ral c ov ale n t m od i f ic a ti on o f DNA i n hu m an s a nd is c h aracterize d by DNA c y t o si n e met hy lati on t ha t occu r s nea rl y e x cl u si v el y at t h e fift h po siti on o f t h e c y t o si n e ri ng i n cy t os i nes p r e ce d i ng gu a n i n e , t h e s o -calle d C p G d i nu cle o ti de .

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Abnormal Gene Silencing

nan nan

L i k e m os t b i o l og i c p r ocess es , t h e no rmal p atter n s o f sile n ci ng ca n b e alte r e d, resu lti ng i n t he deve l op me n t o f d isease states . T hu s , acti v ati on o f g e n es no rm a ll y no t exp r essed, o r sile n ci ng o f a g e n e t h at s hou l d b e e xpr ess ed, can con tri bu t e t o t h e dy sre gu lati on o f g e n e f un cti on t h at c h a r acteri zes cance r and, when sta b l y p rese n t , re p rese n t e p i g e n etic alte r ati ons . Mos t s t ud i es ha ve f o c u se d on t h e sile n ci ng o f no rmall y e xpr ess ed genes. F o r t he pu r p oses o f und ersta nd i ng t h e rati on ale b e h i nd e p i g e n et ic t he r ap y , it i s im po rta n t t o und ersta nd t h e mec h a n isms t h r ough wh ic h su ch s il enc i ng occu r s. A lterati on s i n g e n e e xp ressi on ass o ciate d with e p i g e n et ic changes t ha t g i ve r i se t o a g r o wt h a dv a n ta g e w ou l d b e e xp ect ed t o b e sel ec t ed f o r i n t he hos t tiss u e , lea d i ng t o p r og ressi v e dy sre gu late d grow t h o f t he t u m o r . S uch dys re gu lati on is c o mm on l y ass o ciate d wit h

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Abnormal Gene Silencing

nan nan

i n c r ease s i n p r o m o t e r r eg i on DNA met hy lati on a nd is ass o ciate d wit h r e pr essi ve ch r o m a ti n changes.

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Abnormal Gene Silencing

nan nan

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Abnormal Gene Silencing

nan nan

Abnor m a l de novo DNA m e t hy lati on o f g e n e p r o m o ter C p G isla nd s is a v e ry fr e quen t abno rm a lit y i n v irt u all y all ca n cer t yp es a nd is ass o ciate d w it h a p r ocess t ha t can se r ve a s a n alter n ati v e mec h a n ism f o r l o ss o f t umo r s uppr es so r gene f unc ti on . Alt hough a limite d nu m b er o f classic t u m o r s uppr es so r genes can be a f f ec te d by t h is p r o cess , a p atie n t ’ s i nd i v i du al ca n ce r m ay ha r bo r hund r eds o f s u c h g e n es . W h ic h o f t h ese latter g e n e s a r e dr i ve r s o f cance r , i nd i v i d uall y o r i n g r oup s , v ers u s t ho se w h ic h are p asse ng er s r e fl ec ti ng on l y t he wi d es p rea d effects o f a g l ob al e p i g e n etic a bnor m a lit y i s a l ead i ng ques ti on i n t h e fiel d a nd t h e ta r g et o f m u c h r esea r c h A c l ue t o t he im po rta n ce o f at least g r oup s o f t h e p re v i ou s D NA hyp e r m e t hy l a t ed genes m ay co me fr o m t h e fact t h at a n i no r d i n ate nu m be r of t h em a r e i nvo l ved i n ho l d i ng no rmal em b r yon ic a nd a du lt stem cells i n t h e sel f- r enewa l s t a t e and / o r re nd eri ng s u c h cells refract o r y t o d i f f e r e n ti a ti on cues No rm a ll y , t h ese g e n es are t h e n i n a po ise d e xpr essi on s t a t e and can be i ndu ce d t o b e acti v ate d o r re p resse d as n ee d e d for c h a nges i n ce ll s t a t e Abno rmal p r o m o ter DNA met hy lati on o f s u c h g e n es r e nde r s t he m m o r e r ep r e sse d a nd c ou l d b e a fact o r i n t h e fact t h at ca n ce r s i nev it ab l y exh i b it ce ll popu lati on s wit h e nh a n ce d self-re n ewal or r e fr act or i ness t o f u ll d i f f e r en tiati on.

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Abnormal Gene Silencing

nan nan

Rece n t s t ud i es have a l so sugg este d t h at DNA re g i on s o t h er t h a n pro m o ter CpG i s l ands m ay und e r go c h a ng es o f DNA met hy lati on i n ca n c e r . F or e x am p l e, non–CpG -ri ch se qu e n ces s u rr ound i ng p r o m o ter C p G isla nds, te r me d CpG i s l and sho r es, a re a bno rmall y met hy late d i n ca n cer s a nd m ay b e alte red i n s t e m ce ll popu l a ti on s T hu s , t h e relati v e ca n cer s p ecificit y o f c h a ng es o f DNA m e t hy l a ti on i n m u lti p le C p G re g i on s ma k es re v ersal o f t h ese c hanges by t a r ge ti ng DN A met hy ltra n sferases , t h e e n z y mes t h at catal y ze DNA m e t hy l a ti on, l og ical f o r ca n cer t h era p e u tics . A s a key exa m p l e o f t he p re v i ou s po i n ts , p er h a p s t h e m o st st ud ie d t u m o r s uppr es so r gene f o r p r o m o t e r hyp ermet hy lati on is t h e p16 g e n e , c u rre n tl y d esi gn at ed CDKN2 A , a cyc li n - d e p e nd e n t k i n ase i nh i b it o r t h at f un cti on s i n t h e r e gu l a ti on o f t he phospho r y lati on o f t h e R b p r o tei n. H yp ermet hy lati on ass o ciate d w it h l oss o f exp r ess i on o f t h e CDKN 2 A g e n e h as b ee n f ound to b e on e of t he m os t fr equen t a lterati on s i n n e op lasia b ei ng c o mm on i n t h e l ung, h e ad and neck, g li o m as, c o l o rectal , a nd b reast carci no ma s a nd o t h e r can ce r t ypes. A m e m be r o f t h e same g e n e famil y , p15 o r CDKN 2 B , als o r e gu l a t es Rb and i s s il enc e d i n ass o ciati on wit h p r o m o ter met hy lati on i n ma ny f o rm s o f l euke mi a and i n t h e c h r on ic m y el o i d n e op lasm m y el odysp l as ti c synd r o m e (MDS) . T h ese tw o p re v i ou s c h a ng es are o f m u c h r el evance f o r t he c li n i ca l u ses o f e p i g e n etic t h era p ies d isc u sse d lat e r . A s me n ti oned, m any hund re d s o f g e n es ma y b e i n acti v ate d i n a si ng l e ca n ce r by p r o m o t e r m e t hy l a tio n , p r ov i d i ng po te n tial ta r g ets f o r g e ne r eacti v a t i on us i ng ep i gene ti c t h era p ies . T h e latter re p rese n ts on e o f t he po te n tial ways i n wh i ch ep i gen etic t h era py ma y b e effecti v e: M u lti p le genes a nd g e n e pa t hways, a ll r ep r es se d by c h a ng es i n DNA met hy lati on a nd c hro mati n m od ifi ca ti on, can be reacti v ate d by DNA met hy ltra n sferase i nh i b it ors and h i s t one deace t y lase (HDAC) i nh i b it o rs (HDACi) , t h ere by r est or i ng no rm a l ce ll cyc l e con tr o l , d iffere n tiati on, a nd a pop t o tic si gn alin g . , I n gene r a l , m e t hy late d C p G isla nd s are no t ca p a b le o f t he i n itiati on o f tr ansc ri p ti on un less t h e met hy lati on si gn al ca n b e ov erri dd e n by alte ra ti ons i n f ac t o r s t ha t m odu late c h r o mati n, s u c h as t h e rem ov al o f met hy lat ed cy t os i ne - b i nd i ng pr o tei n s . H o we v e r , re v ersal o f DNA met hy lati on w it h seconda r y c ha ng es i n h ist on e m od ificati on o r d irecte d

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Abnormal Gene Silencing

nan nan

r e v e r sal o f r ep r ess i ve h i s t one mod ificati on s re p rese n t a ta r g et f o r e p i g e netic t h e r a p ies . M o st s t ud i es o f DNA m e t hy lati on, p artic u larl y i n t h e st udy o f ca n ce r , h a v e focused on CpG i s l and p r o m o ter met hy lati on. H o we v e r , a bou t 40 % o f hu ma n genes do no t con t a i n bon a fi d e C p G isla nd s i n t h eir p r o m o ters . The pr ima ry f ocus on CpG i s l and s h as res u lte d fr o m t h e clear d em on strati on t h at C p G-i s l and p r o m o t e r m e t hy lati on p erma n e n tl y sile n ces g e n es bo t h phy si o l og i ca ll y and pa t ho l og icall y i n mammalia n cells . H o we v e r , rece n t work h a s shown co rr e l a ti ons b etwee n tiss u e-s p ecific e xp ressi on a nd met hy lati on o f non - CpG i s l a n d s , i n cl ud i ng, f o r e x am p le , t h e mas p i n g e ne ,

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Abnormal Gene Silencing

nan nan

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Abnormal Gene Silencing

nan nan

a nd as m en ti oned p r ev i ous l y , re g i on s n ear C p G isla nd s , s ugg esti ng t hat

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Abnormal Gene Silencing

nan nan

ma ny a dd iti ona l genes cou l d b e re gu late d, eit h er no rmall y o r a bno rmall y , by c h a nges i n DNA m e t hy l a ti on. An e xc iti ng new a r ea o f D NA met hy lati on researc h i nvo l v es t h e r o le o f t h is c h a nge i n r egu l a ti ng gene e nh a n cers: small DNA re g i on s t h at re gu l ate t h e e xpress i on o f m u lti p l e t a r g et g e n es . T h e p rese n ce o f DNA met hy lati on i n t hese a r eas, wh ic h ca n resi d e c on si d era b le d ista n ces fr om the g e n es t ha t a r e be i ng r egu l a t ed, g e n erall y w o r k s t og et h er wit h h ist on e m od i f ic a ti ons t o m ed i a t e a r ep ressi v e state f o r t h at e nh a n ce r – T h e sta tus of e nh a nce r s i s a l so e m e r g i ng as im po rta n t f o r ca n cer ris k states

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Ch r omatin in Gene Regulation

nan nan

H e r ita b l e gene s il enc i ng i nvo l v es t h e i n ter p la y b etwee n DNA met hy lati on a nd h ist one cova l en t m od ifi c ati on s . C o m p le x es o f p r o tei n s t h at ca n re gulate how nuc l eoso m es a r e pos iti on e d p erf o rm nu cle o s o mal rem od eli ng .

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Ch r omatin in Gene Regulation

nan nan

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Ch r omatin in Gene Regulation

nan nan

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Ch r omatin in Gene Regulation

nan nan

A li nk be t ween cova l en t h ist on e m od ificati on s a nd DNA met hy lati on h as b ee n c l ea rl y es t ab li shed I n t h is i n teracti on, c y t o si n e met hy lati on att r acts m e t hy l a t ed DNA - b i nd i ng p r o tei n s a nd HDACs t o met hy late d C pG sites during ch r o m a ti n co m pac ti on a nd g e n e sile n ci ng . I n a dd iti on, t he DNA m e t hy l a ti on b i nd i ng p rotei n (MBD 2 ) i n teracts wit h t h e nu cle o s o m al r em od eli ng co m p l ex ( NuRD ) a nd d irects t h e c o m p le x t o met hy late d DNA .

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Ch r omatin in Gene Regulation

nan nan

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Ch r omatin in Gene Regulation

nan nan

Th is c o m p l ex a l so b i nds HD ACs a nd h as rece n tl y b ee n i d e n tifie d as a ce n t r al p l aye r f o r t he abno rm a l sile n ci ng o f g e n es ass o ciate d wit h p r o m ote r DNA h y pe rm e t hy l a ti on i n can ce r . T hu s , t h e t h ree p r o cesses o f DNA c y t o si n e m e t hy l a ti on, h i s t one m od ificati on, a nd nu cle o s o mal rem od eli ng a r e i n tim a t e l y li nked, and a lt e rati on s i n t h ese p r o cesses ca n res u lt i n a bnor m a liti es o f gene exp r ess i on i n ca n ce r -rele v a n t g e n es .

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Enzymes Regulating DNA Methylation and Histone Acetylation

nan nan

DNA m e t hy l a ti on i nvo l ves t he c ov ale n t a dd iti on o f a met hy l g r oup t o t he 5′ po siti on o f cy t os i ne. I n m a m m als , t h ree e n z y mes h a v e b ee n s ho w n t o catal y ze t h i s tr ans f e r o f a m e t hy l g r oup fr o m t h e met hy l dono r S-a d e no s y lm e t h i on i ne. Mos t o f t h e met hy ltra n sferase acti v it y p rese n t i n d i f f e r e n ti a t ed ce ll s i s de ri ved fr o m t h e e xp ressi on o f DNMT 1 . T h is e n z y me is t hough t t o be m os t im po rta n t i n mai n tai n i ng DNA met hy lati on p atte rn s f o ll ow i ng DNA r ep licati on a nd t hu s is referre d t o as a mai n te n a nce met hy lt rans f e r ase. Howeve r , t h e e n z y me do es po ssess t h e a b ilit y t o met hy lat e p r ev i ous l y un m e t hy late d DNA se qu e n ces ( d e novo acti v it y ) . I n c on t r ast , t he o t he r enzy m es, DNMT 3 a a nd DNMT 3b, are efficie n t at met hy lati ng p r ev i ous l y un m e t hy late d DNA a nd t hu s are c on si d ere d d e novo met hy lt rans f e r ases. E ach o f these e n z y mes po ssesses a similar catal y tic site a f ac t im po rt an t f o r t he i nh i b iti on o f DNMT e n z y mes by nu cle o si de a n al og s , d i scussed l a t e r i n t h is c h a p te r . DNA m e t hy l a ti on i s c l ose l y ass o ciate d wit h c h a ng es i n t h e h ist on e m od i f ic a ti ons. As p r ev i ous l y d isc u sse d, h ist on e p r o tei n s are t h e ce n tral c o m ponen t s o f t he nuc l eosom e , a nd m od ificati on s o f t h e h ist on e tails o f

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Enzymes Regulating DNA Methylation and Histone Acetylation

nan nan

c or e h is tones a r e assoc i a t ed wit h acti v e o r re p resse d c h r o mati n . Alt hough it is b e yond t he scope o f t h i s c h a p ter t o f u ll y d isc u ss t h e c o m p le x series o f m od i f ic a ti ons t o t he h i s t one tails o f h ist on e H 3 a nd H 4, a few well-c h a r acteri zed m od ifi ca ti ons shou l d b e me n ti on e d t h at are rele v a n t t o t h e r a p ies des i gned t o t a r ge t ep i g e n etic a bno rmalities i n ca n ce r . I n refere nce t o c urr e n tl y i nves ti ga t ed ep i g e n etic t h era p ies , c h a ng es i n h ist on e acet y lat ion a r e of im po rt ance. Ace t y l a ti on o f h ist on es H 3 a nd H 4 at k e y ami no aci d s is ass o ciate d w it h t he ac ti ve ch r o mati n p rese n t at t h e p r o m o ters o f tra n scri bed g e n es , whe r eas t he absence o f h ist on e acet y lati on is ass o ciate d wit h r e pr esse d, s il enced genes Hist on e acet y ltra n sferases (H A T) HDAC s h a v e oppos i ng f unc ti ons t o mai n tai n t h e p r op er le v el o f h ist on e acet y lati on for g e n e exp r ess i on HD ACs s p ecificall y d eacet y late t h e l y si n e r esi du es o f t he h i s t one t a il s, and t h is d eacet y lati on is ass o ciate d wit h c ond e n s a ti on o f nuc l eoso m e po siti on s i n w h at is terme d a cl o se d c h r o m atin for mati on. T h i s scena ri o i s ke y t o tra n scri p ti on al re p ressi on. T h ere are f ou r classes o f HDACs C l ass I H DACs are c h aracterize d by t h eir similarit y to t h e y east Rpd3 HDAC. I n hu ma n s , t h is class o f e n z y mes i n cl ud es HDA C1, 2, 3, a nd 8. T hese HDACs a r e t hough t t o b e ub i qu it ou sl y e xp resse d i n tiss u e t h r oughou t t he bod y . I n c on trast , class II HDACs are similar t o y ea st Hd a 1 a n d i nc l ude HDAC4, 5, 6, 7, 9, a nd 10, a nd t h e y h a v e a g reater d e g r ee of tiss u e spec ifi c it y . C l ass III HDACs are similar t o y east Sir 2 a nd are s et a p a r t fro m t he o t he r c l asses by t h eir d e p e nd e n ce on n ic o ti n ami d e a d e n i ne d i nu cle o ti de ( NAD+ ) as a co f a ct o r . Fi n all y , class IV i n cl ud es HDAC 1 1 Of t he p r ev i ous l y li s t ed HD ACs , class I a nd 2 HDACs h a v e b ee n m ost cl o sel y t ied t o gene s il enc i ng ass o ciate d wit h a bno rmal p r o m o ter DNA hyp e r m e t hy l a ti on . T hese a re bound t o t h e nu cle o s o me rem od eli ng c o m p le x, NuRD . E xpe rim en tal d ecreases i n NURD , after u se o f a DN A d emet hy l a ti ng agen t , can aug me n t reacti v ati on o f ma ny a bno rmall y sile n ce d and DNA hype rm e t hy late d g e n es i n c o l on ca n cer cells .

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Enzymes Regulating DNA Methylation and Histone Acetylation

nan nan

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Enzymes Regulating DNA Methylation and Histone Acetylation

nan nan

Ma n i pu lati on o f t hese HDAC s is und er st udy i n cli n ical trials , wit h a nd w it hou t t he use o f DNA m e t hy ltra n sferase i nh i b it o rs , a nd is d isc u sse d lat e r . Ano t h e r HDAC, SI R T 1 i n t h e class III o f t h ese p r o tei n s , is als o i nvo l v e d w it h g e ne s il enc i ng T h i s d eacet y lase h as b ee n li nk e d t o sile n ci ng o f

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Enzymes Regulating DNA Methylation and Histone Acetylation

nan nan

DNA h y pe rm e t hy l a t ed genes, a nd b l o c k i ng its acti v it y ca n b e ass o ciate d w it h r ea c ti va ti on o f such gene s

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Reversal of Layers of Gene Silencing

nan nan

Th e i n ter ac ti on be t ween DNA met hy lati on a nd HDAC acti v it y a nd r e pr essi ve ch r o m a ti n m a r ks i n mai n tai n i ng a b erra n t sile n ci ng o f hyp e r m e t hy l a t ed genes i n can cer h as t h era p e u tic im p licati on s f o r e p i g e netic t h e r a p ies . E xpe rim en t a l ev i de nce s ugg ests t h at DNA met hy lati on f un cti ons as a do mi nan t even t t ha t s t ab l y esta b lis h es tra n scri p ti on al re p ressi on. Inh i b itio n o f HDAC ac ti v it y al on e , by po te n t a nd s p ecific HDACis , do e s no t g e ne r a ll y r esu lt i n t he r ea cti v ati on o f a b erra n tl y sile n ce d a nd d e n sel y hyp e r m e t hy l a t ed genes i n t umo r cells . I n c on trast , treatme n t wit h HD A C is ca n r eacti va t e dense l y s il enced g e n es if t h e cells are first treate d wit h d emet hy l a ti ng d r ugs, such as 5 -azaciti d i n e . T h e cli n ical im p licati on s o f

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

Reversal of Layers of Gene Silencing

nan nan

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

DNA Methyltransferase Inhibitors

nan nan

Or i g i n all y syn t hes i zed as cy tot ox ic a n timeta bo lite d r ug s i n t h e 1960 s ,

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

DNA Methyltransferase Inhibitors

nan nan

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

DNA Methyltransferase Inhibitors

nan nan

azac y t os i ne nuc l eos i des we r e rec ogn ize d as i nh i b it o rs o f DNA met hy lati on i n t h e ea r l y 1980s. T he i nh i b it o rs 5 -azaciti d i n e ( 5 AC) a nd 2 ′- d e oxy - 5 -azac y ti d i ne i nduced m usc l e, fat , a nd c hond r o c y te d iffere n tiati on i n m ouse em bryo ce ll s, i n assoc i a ti on wit h a re v ersal o f DNA met hy lati on . T he i n c orpor ati on o f azacy t os i ne n u cle o si d es i n t o DNA i n lie u o f c y t o si n e r esi du es was shown t o be asso ciate d wit h i nh i b iti on o f DNMT acti v it y .

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

DNA Methyltransferase Inhibitors

nan nan

E P IG E NETIC ABNORMA L I T IES AND GE NE EXPR E SSION CHANGES IN CAN C ER

DNA Methyltransferase Inhibitors

nan nan