VU Research Portal

Targeted chemoprevention of head and neck cancer

de Boer, D.V.

2021

document version

Publisher's PDF, also known as Version of record

Link to publication in VU Research Portal

citation for published version (APA)

de Boer, D. V. (2021). Targeted chemoprevention of head and neck cancer.

General rights

Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain

• You may freely distribute the URL identifying the publication in the public portal ? Take down policy

If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

E-mail address:

vuresearchportal.ub@vu.nl

Targeting PLK1 as a novel chemopreventive

approach to eradicate preneoplastic mucosal

changes in the head and neck

'9LFN\GH%RHU6DQQH50DUWHQVGH.HPS0DULMNH%XLM]H0DULMNH 6WLJWHUYDQ:DOVXP(OLVDEHWK%ORHPHQD5DOI'LHWULFK&5HQp/HHPDQV 9LFWRU:YDQ%HXVHFKHP%RXGHZLMQ-0%UDDNKXLV5XXG+%UDNHQKRͿ

 

3

INTRODUCTION

:RUOGZLGH DURXQG  FDVHV RI KHDG DQG QHFN VTXDPRXV FHOO FDUFLQRPD +16&&DULVHHDFK\HDUIRUPLQJDERXWRIDOOFDQFHUFDVHV_{+16&&VGHYHORS}

LQ WKH PXFRVDO OLQLQJ RI WKH ODU\Q[ K\SRSKDU\Q[ RURSKDU\Q[ RU RUDO FDYLW\ DQG PRVWLPSRUWDQWULVNIDFWRUVDUHWREDFFRVPRNLQJH[FHVVLYHDOFRKROFRQVXPSWLRQDV ZHOODVLQIHFWLRQZLWKKLJKULVNKXPDQSDSLOORPDYLUXV+393DWLHQWVKDYHD\HDU VXUYLYDOUDWHRIPDLQO\GXHWRWKHKLJKLQFLGHQFHRIORFDOUHODSVHVDQGVHFRQG SULPDU\WXPRUVWKDWDUHGL΀FXOWWRWUHDWFXUDWLYHO\2_. $QLPSRUWDQWSKDVHLQ+16&&GHYHORSPHQWLVWKHIRUPDWLRQRIJHQHWLFDOO\DOWHUHG SUHFDQFHURXVÀHOGV7KHVHÀHOGVGHYHORSLQVHYHUDOVWHSVVWDUWLQJZKHQDPXFRVDO VWHPFHOOXQGHUJRHVPXWDWLRQVDQGRWKHUHSLJHQHWLFDOWHUDWLRQV7KHVHPXWDWLRQVDUH VXEVHTXHQWO\WUDQVIHUUHGWRWKHGDXJKWHUWUDQVLWDPSOLI\LQJDQGGLͿHUHQWLDWLQJFHOOV IRUPLQJ D JHQHWLFDOO\ DOWHUHG FORQDO XQLW ¶SDWFK· LQ WKH PXFRVDO OLQLQJ 7KURXJK IXUWKHUDFFXPXODWLRQRIJHQHWLFFKDQJHVWKHVHSDWFKHVRYHUJURZWKHQRUPDOPXFRVD DQG IRUP ÀHOGV RI JHQHWLFDOO\ DOWHUHG FHOOV WKDW PD\ UHDFK GLPHQVLRQV RI  FP LQ GLDPHWHU'HVSLWHWKHFDQFHUUHODWHGJHQHWLFDOWHUDWLRQVLQWKHVHFHOOVVXFKDVTP53 PXWDWLRQVDQGORVVHVRIWKHFKURPRVRPDODUPVSSDQGSWKHVHSUHQHRSODVWLF ÀHOGVDUHQRW\HWLQYDVLYH2_.

3UHQHRSODVWLF ÀHOGV DUH VRPHWLPHV PDFURVFRSLFDOO\ YLVLEOH DV OHXNRSODNLD ZKLWH OHVLRQVRUHU\WKURSODNLDUHGOHVLRQV_{KRZHYHUPRVWÀHOGVDUHLQYLVLEOHWRWKHQDNHG}

H\H²_{ 2WKHUV GHPRQVWUDWHG WKH SUHVHQFH RI SUHQHRSODVWLF ÀHOGV E\ XVLQJ D GHYLFH}

WKDW GHWHFWV DXWRÁXRUHVFHQFH &KDQJHV LQ DXWRÁXRUHVFHQFH LQGLFDWHG QHRSODVWLF ÀHOGVYDU\LQJLQVL]HLQRIWKHWHVWHG+16&&SDWLHQWV_{. The presence of these}

ÀHOGV IRUPV D KLJK ULVN IRU ORFDO UHODSVHV DQG VHFRQG SULPDU\ WXPRUV HVSHFLDOO\ VLQFHZLWKRXWYLVXDOL]DWLRQWKHVHLPSHUFHSWLEOHSUHQHRSODVWLFFHOOVVXUURXQGLQJWKH WXPRUPD\VWD\EHKLQGZKHQWKHSULPDU\WXPRULVUHVHFWHG²_{,QWULJXLQJO\UHVHFWLRQ}

RI ÀHOGV E\ JXLGDQFH RI DXWRÁXRUHVFHQFH KDG D VLJQLÀFDQW LPSDFW RQ WKH ORFDO UHFXUUHQFHIUHHVXUYLYDO_{7KLVSUHOLPLQDU\REVHUYDWLRQVWUHVVHVWKHXUJHRIDVXLWDEOH}

SUHYHQWLYH WUHDWPHQW WR HUDGLFDWH SUHQHRSODVWLF ÀHOGV ZKHQ GLDJQRVHG E\ HLWKHU FOLQLFDOLQVSHFWLRQDXWRÁXRUHVFHQFHKLVWRORJLFDOH[DPLQDWLRQRUJHQHWLFDQDO\VLV$V WKHVHÀHOGVDUHSUHQHRSODVWLFDQGQRWLQYDVLYHFDQFHUSUHYHQWLYHWUHDWPHQWVVKRXOG EHHͿHFWLYHEXWZLWKOLPLWHGWR[LFLW\5DGLRWKHUDS\LVWKHUHIRUHQRWLQGLFDWHG6XUJLFDO H[FLVLRQ LV DQ RSWLRQ EXW LQ FDVHV ZKHUH YLVLEOH OHVLRQV TXDOLI\ IRU UHVHFWLRQ WKH\ IUHTXHQWO\UHFXU7DUJHWHGWUHDWPHQWDSSURDFKHVDUHOHVVWR[LFWKDQFODVVLFDOF\WRWR[LF DJHQWVDQGVHHPPRVWSURPLVLQJLQWKLVUHVSHFW7RGHYHORSWKHVHGUXJJDEOHJHQH WDUJHWVVKRXOGEHLGHQWLÀHGDVDÀUVWVWHS $SUHUHTXLVLWHIRUWKHLGHQWLÀFDWLRQRISURPLVLQJWDUJHWJHQHVLVWKHDYDLODELOLW\RI SUHQHRSODVWLFFHOOPRGHOV3UHYLRXVO\DSUHQHRSODVWLFFHOOOLQH98SUH6&&0ZDV ABSTRACT

+HDG DQG QHFN VTXDPRXV FHOO FDUFLQRPDV +16&&V DQG ORFDO UHODSVHV WKHUHRI GHYHORSLQSUHQHRSODVWLFÀHOGVLQWKHPXFRVDOOLQLQJRIWKHXSSHUDHURGLJHVWLYHWUDFW 7KHVH ÀHOGV DUH FKDUDFWHUL]HG E\ WXPRUDVVRFLDWHG JHQHWLF FKDQJHV DUH IUHTXHQWO\ G\VSODVWLFDQGRFFDVLRQDOO\PDFURVFRSLFDOO\YLVLEOH&XUUHQWO\QRDGHTXDWHWUHDWPHQW RSWLRQVH[LVWWRSUHYHQWWXPRUGHYHORSPHQW$UUD\EDVHGVFUHHQLQJZLWKDSDQHORI WXPRUOHWKDOVPDOOLQWHUIHULQJ51$VVL51$VLGHQWLÀHG3ROROLNHNLQDVHPLK1DV HVVHQWLDOIRUVXUYLYDORISUHQHRSODVWLFFHOOV,QKLELWLRQRI3/.FDXVHGFHOOGHDWKRI SUHQHRSODVWLFDQG+16&&FHOOVZKLOHSULPDU\FHOOVZHUHKDUGO\DͿHFWHG%RWKVL51$V DQGVPDOOPROHFXOHLQKLELWRUVFDXVHGDVWURQJ*0FHOOF\FOHDUUHVWDFFRPSDQLHG E\IRUPDWLRQRIPRQRSRODUVSLQGOHV,QD[HQRJUDIWHGPRXVHPRGHO3/.LQKLELWLRQ FDXVHG D VLJQLÀFDQW WXPRU JURZWK GHOD\ DQG FXUHV ZKLOH FKHPRUDGLDWLRQ KDG QR HͿHFW7KXV3/.VHHPVWREHDSURPLVLQJWDUJHWIRUFKHPRSUHYHQWLYHWUHDWPHQWRI SUHQHRSODVWLFFHOOVDQGFRXOGEHDSSOLHGWRSUHYHQW+16&&DQGORFDOUHODSVHV

 

3

T

able 1: Re-screen of 319 ‘tumor

-lethal’

siRNAs revealed 98 genes to be essential for the preneoplastic cell line VU-preSCC-M3.

Original genome-wide screen

a Re-screen b SW1573 only a 197 essential siRNAs Overlap a 62 essential siRNAs VU-SCC-120 only a 60 essential siRNAs T o tal a 319 essential siRNAs Hits ( n ) P ercentage Hits ( n ) P ercentage Hits ( n ) P ercentage Hits ( n ) P ercentage SW1573         VU-SCC-120          VU-preSCC-M3         a+LWV LGHQWLÀHG LQ RULJLQDO JHQRPHZLGH VFU HHQV RI WZR WXPRU FH OO OLQHV RULJLQDWLQJ IU RP 6: RU 9 86&& DORQH RU DV RYH UODSSLQJ KLWV RI E RWK FHOO OLQHV 2I Q RWH LQ WKH SU HYLRXV VWXG\ VL51$ KLWV ZHU H LGHQWLÀHG E\ UR EXVW =VFRU H DQDO\VLV ZKLOH LQ WKLV Y DOLGDWLRQ DQDO\VLV D PR UH V WULQJHQW FXWRͿ RI •  FHOOGHDWKZDVFRQVLGHU HGZKLFKZLOOLPSDFWWKHWU XHYDOLGDWLR QSHU FHQWDJHV E5HVFU HHQLQJRI¶WXPRU OHWKDO·VL51$VLQWZRWXPRUFHOOOLQ HVDQGRQHSU HQHRSODVWLFFHOOOLQH JHQHUDWHGE\RXUJURXS_{7KLVFHOOOLQHZDVHVWDEOLVKHGIURPWKHUHVHFWLRQPDUJLQV} RID\HDUROGSDWLHQWZLWKD7D1WXPRULQWKHJORWWLFODU\Q[7KHFHOOVFRQWDLQHG DQRQVHQVHPXWDWLRQLQTP53ORVVRIKHWHUR]\JRVLW\/2+RIFKURPRVRPHDUPVS DQG S DQG ZHUH VKRZQ WR EH SUHQHRSODVWLF LQ DQ RUJDQRW\SLF FHOO FXOWXUH PRGHO +HQFHWKLVFHOOOLQHPRGHODOORZVWHVWLQJRIQRYHOWUHDWPHQWDSSURDFKHV,QUHFHQW \HDUVZHGHYHORSHGPRUHPRGHOVDQGREWDLQHGWKHVHIURPRWKHUUHVHDUFKJURXSVDV ZHOO_.

7KH VHFRQG UHTXLUHPHQW WR GHYHORS DQ DSSURSULDWH WUHDWPHQW LV WKH SUHVHQFH RI VXLWDEOH WDUJHW JHQHV WKDW FDQ EH H[SORLWHG 7KH QXPEHU RI GUXJJDEOH GULYLQJ RQFRJHQHVLQ+16&&LVGLVDSSRLQWLQJO\ORZDQGFRQÀQHGWRPIK3CAPXWDWLRQVLQ OHVVWKDQ_{+HQFHZHUHOLHGRQKLJKWKURXJKSXWIXQFWLRQDOJHQRPLFVDZHOO}

YDOLGDWHGDSSURDFKIRUWKHLGHQWLÀFDWLRQRIHVVHQWLDOJHQHV²_{. Using this approach,}

ZHSUHYLRXVO\LGHQWLÀHGRYHUVL51$VWKDWWDUJHWJHQHVHVVHQWLDOIRUOXQJDVZHOO DVKHDGDQGQHFNFDQFHUFHOOV_{+HUHZHH[DPLQHGWKHVH¶WXPRUOHWKDO·VL51$VLQ} SUHQHRSODVWLFFHOOFXOWXUHVDQGLGHQWLÀHGJHQHVWKDWFDQEHHPSOR\HGWRSRWHQWLDOO\ WDUJHWSUHQHRSODVWLFFHOOV7REHDEOHWRHVWDEOLVKDFOLQLFDOSHUVSHFWLYHVHYHUDODYDLODEOH VPDOOPROHFXOHLQKLELWRUVZHUHWHVWHGDQGWKHXQGHUO\LQJZRUNLQJPHFKDQLVPZDV investigated. RESULTS

Array-based siRNA screening with a ‘tumor-lethal’ library reveals genes essential for preneoplastic cells

7R LGHQWLI\ SRVVLEOHWDUJHWV IRUWKH WUHDWPHQW RISUHQHRSODVWLFÀHOGVZH SHUIRUPHG DQDUUD\EDVHGVFUHHQZLWKSUHYLRXVO\LGHQWLÀHG¶WXPRUOHWKDO·VL51$V_XVLQJ WKHSUHQHRSODVWLFFHOOOLQH98SUH6&&0_{)LJXUH67KHVHVL51$VZHUHRUGHUHG} DVDVXEOLEUDU\DQGDSSOLHGDUUD\EDVHGLQVHSDUDWHZHOOVDVSRROVRIIRXUVL51$V WDUJHWLQJWKHVDPHJHQH7KHRULJLQDOO\VFUHHQHGWXPRUFHOOOLQHV986&&DQG 6:ZHUHDQDO\]HGLQSDUDOOHODVUHIHUHQFH7RLGHQWLI\WKHJHQHVWKDWDUHHVVHQWLDO IRUFHOOVXUYLYDOZHVHWWKHFXWRͿDW•GHFUHDVHLQFHOOYLDELOLW\UHODWLYHWRWKH ZLQGRZ RI WKH SRVLWLYH HJ VL

UBB

 DQG QHJDWLYH FRQWUROV VL&21752/ ,Q FDVH RI6:DQRQVPDOOFHOOOXQJFDQFHU16&/&FHOOOLQHDWRWDORIRXWRI VL51$VPHWWKLVFULWHULRQIRU986&&+16&&ZHLGHQWLÀHGOHWKDO VL51$V7DEOHDQG6&RPELQHGRXWRIVL51$VVKRZHGD OHWKDOHͿHFWLQHLWKHUWKH+16&&DQGRUWKH16&/&FHOOOLQHZLWKWKHXVHGUHODWLYHO\ strict, criterion.

 

3

0 25 50 75 100 siRNA pools siCON

A

siCO N UBB PLK1 -Poo l 0 50 100 C ell v ia bi lit y (% ) VU-preSCC-1640 VU-preSCC-HN472 VU-preSCC-HN433 D34 VU-preSCC-M3 siCON PLK1 -Poo l PLK1 -1 PL K1-2 PLK1 -3 PLK1 -4 0.0 0.5 1.0 Re la ti ve P L K 1 e xp re ss io n _{Primary keratinocytes} VU-preSCC-M3 VU-SCC-120 siCON UB B PLK1 -Poo l PLK1 -1 PLK1 -2 PLK 1-3 PLK1 -4 0 50 100 C el l v ia bil ity (% ) Primary keratinocytes - #50 Primary keratinocytes - #60 VU-preSCC-M3 VU-SCC-120 siCON UBB PLK 1-Pool 0 50 100 C el l vi ab ili ty (% ) Primary keratinocytes - #50 Primary keratinocytes - #60 Primary fibroblasts - #50 Primary fibroblasts - #54 VU-preSCC-M3 VU-SCC-120 * *

B

C

D

E

Ce ll v ia bi lit y (% )

Figure 1:siRNA screening reveals PLK1 as a selective therapeutic target in preneoplastic and

HN-SCC cells. a. 3ORW RI WKH HͿHFW RQ FHOO YLDELOLW\ RI WKH  OHWKDO VL51$ 60$57SRROV DQG FRQWUROV

VFUHHQHGLQ98SUH6&&0%ODFNRSHQGRWVUHSUHVHQWWKHPHGLDQYDOXHVRIWKHSRROVWHVWHGLQ WULSOLFDWHDQGJUH\VTXDUHVDOOVL&21VQHJDWLYHFRQWUROV7KHGRWWHGOLQHLQGLFDWHVVXUYLYDO DQGVL51$SRROVZHUHEHORZWKLVYDOXHb.,QDGGLWLRQWR98SUH6&&0IRXURWKHUSUHQHRSODVWLF FHOOOLQHVDOVRGHPRQVWUDWHG•GHFUHDVHRIFHOOYLDELOLW\DIWHUWUDQVIHFWLRQZLWKWKHPLK1 SMART SRROc3ULPDU\FHOOFXOWXUHVRIQRQWXPRUWLVVXHVDPSOHVZHUHOHVVVHQVLWLYHWRNQRFNGRZQRIPLK1. 98SUH6&&0 DQG 986&& ZHUH LQFOXGHG DV UHIHUHQFH 7KH GLͿHUHQFHV EHWZHHQ ÀEUREODVWV DQGNHUDWLQRF\WHVYHUVXV98SUH6&&0DQG986&&FHOOVZHUHKLJKO\VLJQLÀFDQW P 0DQQ²:KLWQH\8WHVW d. 'HFRQYROXWLRQ RI WKH 60$57SRRO LQ IRXU VHSDUDWH VL51$V VKRZHG DQ DOPRVWLGHQWLFDOSKHQRW\SHIRUWKUHHRXWRIIRXUVHSDUDWHVL51$VIRUDOOFHOOFXOWXUHVWHVWHGe. The 60$57SRRODQGWKHIRXULQGLYLGXDOVL51$VGLUHFWHGDJDLQVWPLK1 UHVXOWHGLQDWOHDVWNQRFN GRZQRIPLK1P51$OHYHOV%DUVUHSUHVHQWWKHPHDQYDOXHVRIWKUHHLQGHSHQGHQWH[SHULPHQWVW\SL FDOO\H[HFXWHGLQWULSOLFDWHLQb, c and d7KHHUURUEDUVUHSUHVHQWWKH6(0%DUVLQÀJXUHe represent PHDQYDOXHVRIWZRLQGHSHQGHQWPHDVXUHPHQWVZLWKHUURUEDUVUHSUHVHQWLQJWKH6'

1H[WZHGHÀQHGWKHSRVVLEOHWR[LFHͿHFWVRI3/.LQKLELWLRQDWOHDVW

in vitro

E\

PLK1

 NQRFNGRZQ LQ QRQPDOLJQDQW FHOOV 2QO\ PLQRU HͿHFWV RQ FHOOXODU YLDELOLW\ ZHUHREVHUYHGDIWHU

PLK1

NQRFNGRZQLQERWKSULPDU\PXFRVDONHUDWLQRF\WHVDQG ÀEUREODVWVRIVHYHUDOKHDOWK\GRQRUV)LJXUHF2IQRWH3/.SURWHLQH[SUHVVLRQ ZDV UHODWLYHO\ ORZ LQ SULPDU\ NHUDWLQRF\WHV )LJXUH 6D ZKLOH WKH GRXEOLQJ WLPH )RU WKH SUHQHRSODVWLF FHOO OLQH 98SUH6&&0 ZH DSSOLHG WKH VDPH FULWHULRQ IRU

OHWKDOKLWLGHQWLÀFDWLRQDQGLGHQWLÀHGOHWKDOVL51$VWKDWWDUJHWDSSDUHQWO\ JHQHVHVVHQWLDOIRUSUHQHRSODVWLFFHOOV)LJXUHD7DEOHDQG62IWKHVHKLWV RULJLQDWHGIURPWKH6:JHQRPHZLGHVFUHHQIURPWKH98 6&& EDVHG VFUHHQ DQG   IURP RYHUODSSLQJ KLWV RI WKH WZR RULJLQDOO\ VFUHHQHGFHOOOLQHV7DEOH:LWKLQWKHVHWRIHVVHQWLDOJHQHVWKDWVKRZHGDOHWKDO SKHQRW\SHLQWKHSUHQHRSODVWLFFHOOVVHYHUDOIXQFWLRQDOFOXVWHUVFRXOGEHLGHQWLÀHG LQFOXGLQJDFOXVWHURIJHQHVUHJXODWLQJWKHPLWRWLFSKDVHRIWKHFHOOF\FOH)LJXUH6 675,1*GDWDEDVHYHUVLRQ

PLK1 seems a potential target for HNSCC treatment and prevention

1H[WDIXUWKHUVHOHFWLRQRISURPLVLQJWDUJHWJHQHVZDVPDGHEDVHGRQWKHH[LVWHQFH RI VPDOO PROHFXOH LQKLELWRUV XVLQJ DYDLODEOH GDWDEDVHV DQG WKH OHWKDO HͿHFW RI WKH VL51$V 2QH RI WKH VL51$V ZLWK WKH VWURQJHVW HͿHFW WDUJHWV

3ROROLNH NLQDVH 



PLK1

:HH[SORUHGWKHIXWXUHRXWORRNRI3/.LQKLELWLRQDVWKHUDSHXWLFVWUDWHJ\ IRUSUHQHRSODVWLFÀHOGV)LJXUH6

PLK1

HQFRGHVDSURWHLQZLWKPXOWLSOHIXQFWLRQV LQWKHUHJXODWLRQRIWKHFHOOF\FOHDQGLVDNQRZQWDUJHWJHQHIRUFDQFHUWUHDWPHQWDQG GUXJJDEOH ZLWK VHYHUDO VPDOO PROHFXOH LQKLELWRUV FXUUHQWO\ WHVWHG LQ FOLQLFDO WULDOV 3/.LVRYHUH[SUHVVHGLQDYDULHW\RIWXPRUW\SHV_{DQGDQXPEHURIVWXGLHVLQGLFDWH}

D FRUUHODWLRQ EHWZHHQ WKH OHYHO RI

PLK1

 H[SUHVVLRQ LQ WXPRUV DQG SURJQRVLV²_.

,QGHHG ZH GHWHFWHG HOHYDWHG OHYHOV RI

PLK1

 H[SUHVVLRQ LQ +16&& WXPRUV E\ DQDO\]LQJPLFURDUUD\P51$SURÀOHVRIDSDQHORI+16&&WXPRUVZLWKPDWFKHG QRUPDOPXFRVDVDPSOHV:HIRXQGKLJKHUH[SUHVVLRQRI

PLK1

LQDOOWXPRUVDPSOHV ZLWKRQDYHUDJHDIROGLQFUHDVHFRPSDUHGWRQRUPDOPXFRVD

P

6WXGHQW

t

WHVW)LJXUH6,QFRQWUDVWWKH51$H[SUHVVLRQOHYHOVRIWKHWZRPRVWFORVHO\UHODWHG 3/. IDPLO\ PHPEHUV

PLK2

and

PLK3

 ZHUH ERWK ORZHU LQ WKH WXPRU VSHFLPHQV DQGIROGUHVSHFWLYHO\

PLK4

LVDOVRH[SUHVVHGDWDKLJKHUOHYHOLQWXPRUV DOWKRXJKZLWKDORZHUIROGLQFUHDVHWKDQ

PLK1

IROG2IQRWHRQO\

PLK1

ZDV LGHQWLÀHGDVDKLWLQWKHLQLWLDOVFUHHQVDQGQRQHRIWKHRWKHUPHPEHUVRIWKHIDPLO\ RISROROLNHNLQDVHV3ONV7KHH[SUHVVLRQGDWDFRQÀUPVWKDW3/.LQKLELWLRQFRXOG LQGHHGVHUYHDVDWKHUDSHXWLFVWUDWHJ\IRU+16&&WUHDWPHQWLQSDWLHQWV

Cell death induced by

PLK1

NQRFNGRZQLVFDQFHUDQGSUHFDQFHUVSHFLÀF

7RDVVHVVWKHPRUHJHQHUDOVXLWDELOLW\RI3/.LQKLELWLRQIRUWUHDWPHQWRISUHQHRSODVWLF ÀHOGV ZH WHVWHG LQ DGGLWLRQ WR 98SUH6&&0 RWKHU SUHQHRSODVWLF FHOO PRGHOV JHQHUDWHGE\XVRUE\RWKHUV_{7DEOH651$LPHGLDWHG}

_PLK1

_{NQRFNGRZQKDG}

WKHVDPHHͿHFWRQFHOOYLDELOLW\LQWKHVHPRGHOVZLWKDWRGHFUHDVHLQYLDEOH FHOOVFRPSDUHGWRWKHQHJDWLYHFRQWURO)LJXUHE

 

3

GSK461364 (μM) 10-4 10-2 100 0 50 100 Ce ll v ia b il it y ( %

) Primary keratinocytes - #50Primary keratinocytes - #60 VU-preSCC-M3 VU-SCC-120 UM-SCC-22A 10-4 10-2 100 0 50 100 BI6727 (μM) C e ll v ia b il it y (%

) Primary keratinocytes - #50Primary keratinocytes - #60 VU-preSCC-M3 VU-SCC-120 UM-SCC-22A B 10-4 10-2 100 102 0 50 100 ON-01910 (μM) C e ll v ia b il it y (% ) VU-SCC-120 VU-preSCC-M3 Primary keratinocytes - #50 A 0.010.0 5 0.10.5 12.5 57.5 10 15 20 25 0 50 100 siRNA (nmol) Ce ll v ia b il it y ( % ) Primary keratinocytes VU-preSCC-M3 C HMN-214 (μM) 10-4 10-2 100 102 0 50 100 Ce ll v ia b il it y ( % ) VU-SCC-120 VU-preSCC-M3 Primary keratinocytes - #50

Figure 2: Selective PLK1 inhibitors show in vitro a therapeutic window between (pre)cancerous cells and primary cells. a.7UHDWPHQWRIFHOOFXOWXUHVZLWK21WRSDQG+01ERWWRP

UHVXOWHG LQ VLPLODU UHVSRQVHV RI SULPDU\ SUHQHRSODVWLF DQG WXPRU FHOOV ZLWK RQO\ D PLQRU WKHUD SHXWLFZLQGRZb.7UHDWPHQWZLWKWZRRWKHUVPDOOPROHFXOHLQKLELWRUV%,WRSDQG*6. ERWWRPVKRZHGDODUJHGLͿHUHQFHEHWZHHQSULPDU\FHOOVYHUVXVSUHQHRSODVWLFDQGWXPRUFHOOVc. As DUHIHUHQFHRIWKHHͿHFWRI3/.LQKLELWLRQE\VPDOOPROHFXOHLQKLELWRUVZHDQDO\]HGDVHULDOGLOXWLRQ range of the PLK160$57SRROVL51$VIURPWLOOQPRO7KHVL&21YDOXHVZHUHVHWDW ZKHUHDVWKHUBBFRQWUROVKRZHGPRUHWKDQUHGXFWLRQRIFHOOYLDELOLW\QRWVKRZQ$OOVPDOOPR OHFXOHJUDSKVLQSDQHOVa and bUHSUHVHQWPHDQYDOXHVRIWKUHHLQGHSHQGHQWH[SHULPHQWVSHUIRUPHG ZLWKWULSOLFDWHVSHUH[SHULPHQWDQGHUURUEDUVLQGLFDWLQJWKH6(03DQHOcUHSUHVHQWVPHDQYDOXHVRI WKUHHLQGHSHQGHQWPHDVXUHPHQWVZLWKHUURUEDUVUHSUHVHQWLQJWKH6(0

G2 / M phase arrest and improper spindle formation after PLK1 inhibition

7RH[SORUHWKHFHOOXODUHͿHFWVRI3/.LQKLELWLRQRQ+16&&DQGSUHFDQFHUPXFRVDO FHOOV 806&&$ DQG 98SUH6&&0 ZHUH H[SRVHG WR %,  ۚ0 RU *6.  ۚ0 DQG DIWHU  KRXUV FHOO F\FOH SURÀOHV ZHUH DQDO\]HG XVLQJ ÁRZF\WRPHWU\,QERWKFDVHVD*0DUUHVWZDVVHHQLQDSSUR[LPDWHO\RIWKH WXPRU DV ZHOO DV WKH SUHQHRSODVWLF FHOOV ,Q DGGLWLRQ ZH QRWHG D VPDOO LQFUHDVH LQ SRO\SORLG\ FHOOV ZLWK D '1$ FRQWHQW ! 1 )LJXUH D DQG 6 ,Q FRQWUDVW WR WKH WXPRUDQGSUHQHRSODVWLFFHOOVSULPDU\PXFRVDONHUDWLQRF\WHVVKRZHGRQO\DPRGHVW *0DUUHVWZKHQWUHDWHGZLWKWKHVDPHFRQFHQWUDWLRQVRI%,DQG*6. )LJXUH D 7KLV LV LQ OLQH ZLWK WKHLU LQVHQVLWLYLW\ WR VL51$ PHGLDWHG NQRFNGRZQ ZDVFRPSDUDEOHDWOHDVWLQWKHÀUVWSDVVDJHV7DEOH6'HFRQYROXWLRQRIWKHPLK1

60$57SRRO WR IRXU GLVWLQFW VL51$V WDUJHWLQJ GLͿHUHQW VHTXHQFHV RI WKH P51$ VKRZHGVLJQLÀFDQWDQGJHQHUDOO\VLPLODUHͿHFWVRQFHOOYLDELOLW\2QO\VL51$ZDV OHVVHͿHFWLYH)LJXUHG6LQFHRXWRIGLVWLQFWVL51$VVKRZHGD!GHFUHDVH RI FHOO YLDELOLW\ IRU 98SUH6&&0

PLK1

 ZDV FRQVLGHUHG D ERQD ÀGH WDUJHW .QRFNGRZQDQDO\VLVE\57T3&5GHPRQVWUDWHGDGHFUHDVHLQP51$H[SUHVVLRQRI DSSUR[LPDWHO\)LJXUHH,QDGGLWLRQWKHSURWHLQOHYHOVDOVRGHFUHDVHGE\ PRUHWKDQ)LJXUH6E

Potency of small molecule PLK1 inhibitors in vitro

6HYHUDOVPDOOPROHFXOHLQKLELWRUVRI3/.DUHEHLQJHYDOXDWHGLQFOLQLFDOWULDOVDQGZH VHOHFWHGIRXURIWKHVHFRPSRXQGVWRWHVWRQWXPRU986&&DQG806&&$ DQGSUHQHRSODVWLF98SUH6&&0FHOOOLQHVDVZHOODVRQSULPDU\NHUDWLQRF\WHV 7ZRRIWKHVHGUXJVLH%,9RODVHUWLEDQG*6.DUHSRWHQWFRPSHWLWLYH LQKLELWRUVRI$73ELQGLQJ215LJRVHUWLELVDQRQFRPSHWLWLYH$73LQKLELWRU DQG+01LVEHOLHYHGWRLQWHUIHUHZLWKWKHVSDWLDOGLVWULEXWLRQRI3/.25_{6WULNLQJO\}

the in vitro FHOOVXUYLYDOSURÀOHVDIWHUWUHDWPHQWZLWKWKHVHIRXUVPDOOPROHFXOHLQKL ELWRUVGLͿHUHGPDUNHGO\UHVXOWLQJLQWZRW\SHVRIUHVSRQVHV21DQG+01 VWURQJO\GHFUHDVHGYLDELOLW\RIWXPRUSUHQHRSODVWLFDQGSULPDU\FHOOV)LJXUHD,Q FRQWUDVW %, DQG *6. ZHUH OHVV WR[LF WR SULPDU\ NHUDWLQRF\WHV FRPSDUHG WRWKHWXPRUDQGSUHQHRSODVWLFFHOOV)LJXUHE6LQFHQRWDOOLQKLELWRUVVKRZHGWKH VDPHHͿHFWZLWKUHVSHFWWRWKHSURÀOHDQGZLQGRZEHWZHHQSUHQHRSODVWLFDQGQRUPDO NHUDWLQRF\WHVDGLOXWLRQUDQJHRIWKHPLK1VL51$60$57SRROZDVPDGHWRFRPSDUH WKHVPDOOPROHFXOHDQGVL51$GRVHUHVSRQVHFXUYHV)LJXUHF7KHGRVHUHVSRQVH FXUYHVRI%,DQGSDUWLFXODUO\*6.VKRZHGWKHFORVHVWUHVHPEODQFHWRWKH VL51$SURÀOHZKLFKVXJJHVWVWKHKLJKHVWWDUJHWVSHFLÀFLW\

 

3

7R FRQÀUP WKH GLUHFW LQYROYHPHQW RI 3/. LQ WKH REVHUYHG FHOO F\FOH DUUHVW ZH DOVR WHVWHG FHOO F\FOH GLVWULEXWLRQ SDWWHUQV DIWHU 51$LPHGLDWHG NQRFNGRZQ )ORZ F\WRPHWU\DQDO\VLVRI98SUH6&&0DIWHUWUDQVIHFWLRQZLWKWKHPLK160$57SRRO UHVXOWHGLQDQDUUHVWLQ*0FRPSDUDEOHWRWKDWRIWKHVPDOOPROHFXOHLQKLELWRUV )LJXUHE

1H[W ZH WHVWHG ZKHWKHU 3/. LQKLELWLRQ LQ SUHQHRSODVWLF FHOOV DͿHFWV VSLQGOH IRUPDWLRQ LQ D VLPLODU ZD\ DV ZDV SUHYLRXVO\ GHPRQVWUDWHG IRU WXPRU FHOOV_.

'LYLGLQJXQWUHDWHGSUHQHRSODVWLFFHOOVDOOVKRZHGQRUPDOVSLQGOHDVVHPEO\PRQR SRODU VSLQGOHV ZHUH REVHUYHG LQ  RXW RI  FRXQWHG FHOOV  0RVW FHOOV WUHDWHG ZLWK*6.GLGQRORQJHUDVVHPEOHSURSHUVSLQGOHVPRQRSRODUVSLQGOHVZHUH REVHUYHGLQRIFRXQWHGFHOOVLQWKHSUHVHQFHRIWKHLQKLELWRUP )LVKHU ([DFW WHVW )LJXUH F 7KHVH UHVXOWV LQGLFDWH LPSURSHU VSLQGOH IRUPDWLRQ LQ 98SUH6&&0FHOOVE\3/.LQKLELWLRQLGHQWLFDODVREVHUYHGLQWXPRUFHOOV

3UHFOLQLFDODVVHVVPHQWRI3/.LQKLELWRUVGHPRQVWUDWHVHͿFDF\LQD+16&& animal model

$VGHVFULEHGDERYHHOHYDWHGH[SUHVVLRQOHYHOVRIPLK1LQSDWLHQWPDWHULDOVXSSRUWV WKHSRWHQWLDORI3/.DVWKHUDSHXWLFWDUJHW7RH[SORUHWKLVLQDPRUHFOLQLFDOUHOHYDQW VHWWLQJ ZH XVHG D +16&& PRXVH PRGHO WUHDWHG ZLWK WKH WZR PRVW SURPLVLQJ LQKLELWRUVDFFRUGLQJWRRXU

in vitro

GDWD%,DQG*6.)LUVWZHGHWHUPLQHG WKHPD[LPXPWROHUDEOHGRVH07'GDWDQRWVKRZQ1H[WPLFHEHDULQJ806&& $[HQRJUDIWWXPRUVZHUHWUHDWHGZLWKWKHWZRVHOHFWHGLQKLELWRUV7DEOH6EDVHG RQ WKH SLORW 07' GDWD ZH REWDLQHG DQG HͿHFWV ZHUH FRPSDUHG WR VROYHQW DQG FKHPRUDGLDWLRQSURWRFROV,QJHQHUDOWUHDWPHQWZDVWROHUDWHGZHOOIRU3/.LQKLELWRUV DVMXGJHGE\FOLQLFDOVLJQVDQGERG\ZHLJKWFKDQJHV)LJXUHD2QHPRXVHLQWKH *6.WUHDWHGJURXSVKRZHGPRUHZHLJKWORVVDQGKDGWREHGLVFRQWLQXHGIURP WUHDWPHQWDQGZDVHXWKDQL]HGDFFRUGLQJKXPDQHHQGSRLQWFULWHULDGD\EXWZDV NHSWLQWKHGDWDDQDO\VLV 7UHDWPHQWUHVXOWHGLQERWKJURXSVLQDVLJQLÀFDQWUHGXFWLRQRIWXPRUYROXPHZKHQ FRPSDUHGWRWKHFRQWUROJURXSDQGDOVRWRWKHJURXSWUHDWHGZLWKUDGLRWKHUDS\DQG FLVSODWLQ

P

DWGD\6WXGHQW

t

WHVW)LJXUHE6XUYLYDOEDVHGRQWKHWLPH WRUHDFKIRXUWLPHVWKHLQLWLDOWXPRUYROXPH[,79VLJQLÀFDQWO\LQFUHDVHG

P

 /RJUDQNWHVWIRUERWKJURXSVWUHDWHGZLWKDVPDOOPROHFXOHLQKLELWRUFRPSDUHG WRVROYHQWZKLOHFKHPRUDGLDWLRQGLGQRWVKRZEHQHÀFLDOHͿHFWV)LJXUHF,QWKH %,JURXSZHREVHUYHGRQHOLNHO\FXUHZKLOHLQWKH*6.WUHDWHGJURXSWZR PLFHVKRZHGDFRPSOHWHUHVSRQVHSURYLGLQJFXUHVLQRXWRIWUHDWHGPLFH 7KHVH UHVXOWV VXJJHVW WKDW SULPDU\ FHOOV KDYH PHFKDQLVPV WR UHVFXH SURSHU FHOO

GLYLVLRQZLWKUHGXFHGDFWLYLW\RI3/.DQGWKHUHE\FLUFXPYHQWWKH*0DUUHVWD FKDUDFWHULVWLFWKDWDSSDUHQWO\KDVEHHQORVWGXULQJFDUFLQRJHQHVLV GSK461364 Untreated VU-preSCC-M3

A

B

C

0% 20% 40% 60% 80% 100% M3 M3 22A 22A PK PK Di st ri but ion ( % of to ta l) BI6727 >4N G2/M S G1 0.25 μM BI6727 - + - + - + 0% 20% 40% 60% 80% 100% M3 M3 22A 22A PK PK Distrib utio n (% of to ta l) GSK461364 >4N G2/M S G1 0.07 μM GSK461364 - + - + - + 0% 20% 40% 60% 80% 100%

Untransfected siCON PLK1-Pool

Di st ribut io n of to ta l (%) VU -preSCC -M3 >4N G2/M S G1 Transfection condition Figure 3: 3/.LQKLELWLRQLQGXFHVD*0DUUHVWLQSUHFDQFHURXVFHOOVWRJHWKHUZLWKORVVRIEL-polar spindle formation. a.98SUH6&&0DQG806&&$DVDUHIHUHQFHZHUHH[SRVHGWR%,

DQG*6.7KLVUHVXOWHGLQDVWURQJ*0DUUHVWIRUERWKFHOOOLQHV3ULPDU\NHUDWLQRF\WHV3. VKRZHGDOHVVSURQRXQFHG*0DUUHVWDIWHUGUXJWUHDWPHQW%DUVUHSUHVHQWWKHSHUFHQWDJHRIFHOOV LQDFHUWDLQSKDVHDVLQGLFDWHG0HDQSHUFHQWDJHVRIWKUHHLQGHSHQGHQWH[SHULPHQWVDUHVKRZQ'DWD ZDVDQDO\]HGZLWKWKH3RLVVRQHTXDWLRQDQGGHPRQVWUDWHGDVLJQLÀFDQWGLͿHUHQFHEHWZHHQWKHFHOO OLQHVDQGWKHSULPDU\FHOOVIRUERWK*6.DQG%,P b.6LPLODUDVWRWKHLQKLEL WRUV98SUH6&&0FHOOVWUDQVIHFWHGZLWKWKHVL51$60$57SRROGLUHFWHGDJDLQVWPLK1XQGHUJRD *0DUUHVW%DUVUHSUHVHQWWKHSHUFHQWDJHVRIFHOOVVLPLODUDVLQGLFDWHGLQa. c.98SUH6&&0FHOOV ZHUHWUHDWHGZLWKۚ0*6.DQGYLVXDOL]DWLRQRIնWXEXOLQVKRZHGGLVRUJDQL]HGPRQRSR ODUVSLQGOHVLQDOPRVWDOOFHOOV,QFRQWUDVWXQWUHDWHGFHOOVGLVSOD\HGRQO\QRUPDOELSRODUDUUDQJHG VSLQGOHV5HSUHVHQWDWLYHSLFWXUHVRIXQWUHDWHGDQGWUHDWHGFHOOVDUHVKRZQVFDOHEDUVLQGLFDWHۚP

 

3

DISCUSSION &DQFHULQWKHKHDGDQGQHFNPD\EHSUHFHGHGE\YLVLEOHRUPDFURVFRSLFDOO\LQYLVLEOH OHVLRQVDQGDVLQGLFDWHGLQSUHYLRXVVWXGLHVWKHVHSUHQHRSODVWLFOHVLRQVDUHDQLP SRUWDQWVRXUFHIRUSULPDU\WXPRUVLQWKHJHQHUDOSRSXODWLRQDQGUHODSVHVLQWUHDWHG HNSCC patients²_{$QHͿHFWLYHWUHDWPHQWRIWKHVHÀHOGVZRXOGEHRIJUHDWEHQHÀW} EXWDVUHVXOWRIWKHLUSUHQHRSODVWLFQDWXUHRQO\WUHDWPHQWVZLWKDORZWR[LFLW\SURÀOH DUHDFFHSWDEOH8QIRUWXQDWHO\WKHUHDUHQRVXLWDEOHWKHUDSHXWLFVWUDWHJLHVWRHUDGLFDWH WKHVHÀHOGV9LVLEOHOHVLRQVFDQEHWUHDWHGE\VXUJHU\RUODVHUEXWH΀FDF\RIWKHVH PRGDOLWLHVLVTXHVWLRQDEOH_{DVWKHOHVLRQVDUHODUJHUWKDQYLVLEOHDQGIUHTXHQWO\UHFXU} 9HU\UHFHQWO\ÁXRUHVFHQFHJXLGHGUHVHFWLRQVVKRZHGSURPLVLQJUHVXOWVDQGSURYLGHG WKH SURRI RI FRQFHSW WKDW DGHTXDWH WUHDWPHQW RI ÀHOGV ZLOO UHGXFH WXPRU EXUGHQ_.

7R LGHQWLI\ WKHUDSHXWLF WDUJHWV IRU WKHVH SUHFDQFHURXV ÀHOGV ZH XVHG DUUD\EDVHG VFUHHQLQJ RI SUHYLRXVO\ LGHQWLÀHG VL51$V WKDW DUH OHWKDO LQ WXPRU FHOO OLQHV DQG LGHQWLÀHGVL51$VWKDWHUDGLFDWHSUHQHRSODVWLFFHOOVLQFXOWXUH)RUFRPSDULVRQZH DOVR UHWHVWHG WKH WZR RULJLQDO WXPRU FHOO OLQHV WKDW ZHUH SUHYLRXVO\ VFUHHQHG DQG PDQ\RIWKHKLWVFRXOGEHFRQÀUPHG)RUERWKFHOOOLQHVPRUHWKDQKDOIRIWKHWHVWHG VL51$VVKRZHGDVLJQLÀFDQWHͿHFWZLWKWKHUHODWLYHO\VWURQJFULWHULRQRI•FHOO GHDWK,QJHQHUDOWKHSUHQHRSODVWLFFHOOVVHHPWREHOHVVVHQVLWLYHWRNQRFNGRZQZLWK WKHVH ¶WXPRUOHWKDO· VL51$V VLQFH RQO\  RI WKH VL51$V JDYH ULVH WR VLJQLÀFDQW F\WRWR[LFLW\LQWKHVHFHOOV7KLVVXJJHVWVWKDWWKHODUJHUPXWDWLRQDOEXUGHQLQWXPRU FHOOV PDNHV WKHP PRUH YXOQHUDEOH IRU NQRFNGRZQ RI VSHFLÀF JHQHV DQ LQWULJXLQJ REVHUYDWLRQ*HQHUDOO\SUHQHRSODVWLFFXOWXUHVVKRZOHVVJHQHWLFFKDQJHVWKDQWXPRU FHOOOLQHV_.

,Q WKLV VWXG\

PLK1

 ZDV VHOHFWHG DV SURPLVLQJ WDUJHW JHQH VLQFH LWV VXSSUHVVLRQ UHVXOWHGLQSURIRXQGJURZWKLQKLELWLQJHͿHFWVLQERWKWXPRUDQGSUHQHRSODVWLFFHOOV EXWPXFKOHVVLQSULPDU\FHOOV7KHIDFWWKDWPLK1LVIRXQGWREHRYHUH[SUHVVHGLQ +16&&VSHFLPHQDQGWKDWLWDOVRVHHPVWREHRYHUH[SUHVVHGLQQHRSODVWLFFHOOPRGHOV

in vitro

DGGVXSWRWKHFRQFHSWRI3/.DVDSURPLVLQJDQGVHOHFWLYHWDUJHW,QWKH SDVWRWKHUVGHPRQVWUDWHGWKHVHHOHYDWHGOHYHOVDVZHOODQGHYHQVKRZHGWKDWDKLJK PLK1H[SUHVVLRQOHYHOLVDVVRFLDWHGZLWKDSRRUSURJQRVLVDQGDORZHURYHUDOOVXUYLYDO time_{7KHSURWHLQHQWHUHGWKHVSRWOLJKWDVWDUJHWPROHFXOHIRUVHYHUDOWXPRUW\SHV} DQGRXUGDWDVXSSRUWWKHLPSRUWDQWUROHRI3/.LQ+16&&FHOOVDVZHOO+HUHZH VKRZWKDWLWPD\VHUYHDVWKHUDSHXWLFWDUJHWLQSUHQHRSODVWLFFHOOVOLNHZLVHDWRWDOO\ QRYHOFRQFHSW

3ON SURWHLQV DUH LPSRUWDQW UHJXODWRUV RI WKH FHOO F\FOH DQG FHOO GLYLVLRQ DQG 3/. LQ SDUWLFXODU SOD\V FULWLFDO UROHV %RWK WKH DFWLYDWLRQ RI 3/. E\ SRVWWUDQVODWLRQDO PRGLÀFDWLRQV LWV FHOOXODU ORFDOL]DWLRQ DQG WKH RYHUDOO H[SUHVVLRQ OHYHOV GHWHUPLQH WKHSUHFLVHUROHGXULQJWKHGLͿHUHQWSKDVHVRIWKHFHOOF\FOHLQFOXGLQJPLWRWLFHQWU\ 0 10 20 30 40 0 200 400 600 800 1000 Days Rel a ti v e t u m o r v o lu m e ( % ) _Solvent RT/Plat BI6727 GSK461364 0 10 20 30 40 80 90 100 110 120 Days Bo d y w e ig h t ( % ) Solvent RT/Plat BI6727 GSK461364

A

B

Tu m o r < 4 x I T V ( % ) Solvent RT/Plat BI6727 GSK461364 0 20 40 60 80 100 0 50 100 Days P<0.03

C

Figure 4: (ͿFDF\ RI 3/. LQKLELWRUV LQ D +16&& [HQRJUDIW PRGHO D 5HODWLYH FKDQJHV LQ ERG\

ZHLJKWVKRZHGWROHUDEOHZHLJKWORVVHVDIWHUWUHDWPHQW$IWHUWUHDWPHQWPLFHUHFRYHUHGTXLFNO\WR WKHLULQLWLDOZHLJKWDQGJDLQHGDGGLWLRQDOZHLJKWb.6LJQLÀFDQWVXSSUHVVLRQRIWXPRUJURZWKUHVXOWV IURPWKHWUHDWPHQWZLWKHLWKHU*6.RU%,,QFDVHRI*6.WZRFRPSOHWHUHVSRQVHV ZHUH REVHUYHG ZKHUH %, VKRZHG RQH PRXVH ZLWK D OLNHO\ FXUH DW ERWK VLWHV %RWK LQKLELWRUV ZHUHVLJQLÀFDQWO\PRUHHͿHFWLYHWKDQWKHVROYHQWDVZHOODVWKHFKHPRUDGLDWLRQSURWRFROP  6WXGHQWtWHVW%RWKJUDSKVa and bUHSUHVHQWPHDQYDOXHVSHUJURXSn DQGHUURUEDUVLQGLFDWH the SEM. c.6XUYLYDOFXUYHVEDVHGRQWKHWLPHXQWLODQLQGLYLGXDOWXPRUUHDFKHVIRXUWLPHVWKHLQLWLDO WXPRUYROXPH[,79%RWK*6.DQG%,VKRZHGDVLJQLÀFDQWLPSURYHPHQWRIVXUYLYDO P DQGUHVSHFWLYHO\/RJUDQNWHVW

 

3

PRGHOVZHXVHGD+16&&DQLPDOPRGHOWRGHPRQVWUDWHWKHFOLQLFDODSSOLFDELOLW\ ZKLFKZDVFOHDUO\GHPRQVWUDWHG%RWKLQKLELWRUVVKRZHGDVLJQLÀFDQWGHOD\RIWXPRU JURZWK DQG HYHQ  FXUHV RXW RI  PLFH IRU *6. DQG RQH FXUHG PRXVH IRU %,7KHVHWZRLQKLELWRUVDUHLQWHUHVWLQJRSWLRQVIRUFKHPRSUHYHQWLYHWUHDWPHQWRI KLJKULVNSUHQHRSODVWLFÀHOGVEXWDOVRIRU+16&&SDWLHQWV*LYHQWKHWR[LFLW\SURÀOH RI WKHVH LQKLELWRUV VHOHFWLRQ RI SDWLHQWV DW KLJK ULVN IRU PDOLJQDQW WUDQVIRUPDWLRQ VHHPVUHTXLUHG

7KHUHLVRQHSRWHQWLDOXQGHVLUDEOHDVSHFWRI%,WKDWUHTXLUHVFOLQLFDOHYDOXDWLRQ DWKLJKHUGRVHVFHOOYLDELOLW\LQFUHDVHG

in vitro

)LJXUHE([DPLQDWLRQRIFHOOF\FOH GLVWULEXWLRQLQWKHSUHVHQFHRIWKHVHKLJKHUFRQFHQWUDWLRQV!ۚ0UHYHDOHGWKDWWKH DUUHVWLQWKH*0SKDVHDOVRGLPLQLVKHGVXJJHVWLQJWKDWOHVVFHOOVVXͿHUIURP3/. LQKLELWLRQGDWDQRWVKRZQ:HDVVXPHWKDWWKLVLVGXHWRRͿWDUJHWHͿHFWVRQNLQDVHV WKDWLQWHUIHUHZLWKWKHHͿHFWVRI3/.LQKLELWLRQDOWKRXJKLWZDVVKRZQSUHYLRXVO\ WKDW  ۚ0 %, GLG QRW KDYH HͿHFWV RQ D SDQHO RI  NLQDVHV_{ 1RQHWKHOHVV}

PXOWLSOHRWKHUFDQGLGDWHNLQDVHVKDYHQRWEHHQWHVWHGDQGVRPHPD\EHDFWLYHLQWKH PROHFXODUPHFKDQLVPVWKDWOHDGWRFHOOGHDWKDIWHU3/.LQKLELWLRQ ,QFRQFOXVLRQ3/.LVDSURPLVLQJGUXJWDUJHWIRUFKHPRSUHYHQWLYHWULDOVWRHUDGLFDWH KLJKULVNSUHQHRSODVWLFÀHOGVDQGWRSUHYHQWWXPRUVLQOHXNRSODNLDSDWLHQWVDQGORFDO UHODSVHVLQWUHDWHG+16&&SDWLHQWV7RRXUNQRZOHGJHZHDUHWKHÀUVWWRUHSRUWRQD QRYHOWDUJHWHGFKHPRSUHYHQWLRQDSSURDFKIRUPXFRVDOSUHQHRSODVWLFFKDQJHVEDVHG RQDUDWLRQDOGHVLJQ

MATERIALS AND METHODS Patient material

&ROOHFWLRQDQGFXOWXUHRISDWLHQWPDWHULDOZDVDSSURYHGE\WKH,QVWLWXWLRQDO5HYLHZ %RDUG RI WKH 98 8QLYHUVLW\ 0HGLFDO &HQWHU SURWRFRO  7LVVXH VDPSOHV RI SDWLHQWVZLWK+16&&ZHUHFROOHFWHGDIWHULQIRUPHGFRQVHQW8VHRIUHVLGXDOWLVVXH RI VXUJLFDO QRQWXPRU VSHFLPHQV ZDV FDUULHG RXW DFFRUGLQJ WR WKH JXLGHOLQHV IRU DQDO\]LQJKXPDQVDPSOHVRIWKH'XWFK0HGLFDO6FLHQWLÀF6RFLHWLHVZZZIHGHUDRUJ

Cell lines

3ULPDU\ FHOOV ZHUH FROOHFWHG IURP H[FLVHG XYXODV RI KHDOWK\ DGXOWV DV SUHYLRXVO\ GHVFULEHG_{3ULPDU\ÀEUREODVWVZHUHFXOWXUHGLQ'XOEHFFRV0RGLÀHG(DJOH0HGLXP}

'0(0 /RQ]D ZLWK  IHWDO FDOI VHUXP /RQ]D DQG  PPRO/ /JOXWDPLQH /RQ]D 7KH SULPDU\ RURSKDU\QJHDO NHUDWLQRF\WHV ZHUH FXOWXUHG LQ VHUXPIUHH .*0PHGLXP.*06)0*LEFRVXSSOHPHQWHGZLWK%6$03%LRPHGLFDOV  PJ ERYLQH SLWXLWDU\ H[WUDFW *LEFR  ۚJ KXPDQ UHFRPELQDQW (*) *LEFR FHQWURVRPH PDWXUDWLRQ DQG ELSRODU VSLQGOH IRUPDWLRQ_{ 7KH RWKHU 3ON IDPLO\}

PHPEHUVVHHPWRKDYHDOHVVLPSRUWDQWUROHDVLQGLFDWHGE\WKHLUH[SUHVVLRQSURÀOHV LQ+16&&WXPRUV0RUHRYHULQWKHRULJLQDOJHQRPHZLGHVFUHHQSHUIRUPHGRQ98 6&&RQO\VL51$VVLOHQFLQJ

PLK1

JDYHULVHWRDOHWKDOSKHQRW\SHZKLFKZDVQRW REVHUYHGIRUVL51$VWDUJHWLQJ

PLK2

,

3

or

4

18_.

,Q D FOLQLFDO VHWWLQJ LQKLELWLRQ RI 3/. PLJKW EH XVHIXO IRU ERWK +16&& WXPRU WUHDWPHQWDVZHOODVSUHYHQWLRQRIWXPRUVDQGORFDOUHODSVHV_{,QSDUWLFXODUSDWLHQWV}

ZKRDUHDWKLJKULVNIRUGHYHORSLQJKHDGDQGQHFNFDQFHUDQGORFDOUHODSVHVFRXOG EHHQUROOHGLQIXWXUHWULDOV5LVNDVVHVVPHQWFDQEHEDVHGRQWKHSUHVHQFHRIJHQHWLF DOWHUDWLRQVLQYLVLEOHOHVLRQVRUWKHVXUJLFDOPDUJLQVIRULQVWDQFHORVVHVRIFKURPRVRPH DUPV S DQG S_{ 2XU GDWD VXJJHVWV WKDW WUHDWPHQW RI YLVLEOH OHXNRSODNLD OHVLRQV}

PLJKWEHVXFFHVVIXODVZHOO:LWKDQDQQXDOWUDQVIRUPDWLRQUDWHRISHU\HDUWKH SUHVHQFHRIYLVLEOHOHXNRSODNLDOHVLRQVGRHVQRWGLUHFWO\OHDGWRDQLPPHGLDWHFDQFHU risk_{EXWDIWHUORQJHUIROORZXSRIWKHVHSDWLHQWVPD\GHYHORSFDQFHU7RXVH} 3/.LQKLELWLRQLQDSUHYHQWLYHWUHDWPHQWVHWWLQJRQO\SDWLHQWVZLWKSUHFDQFHURXV FKDQJHVDWKLJKULVNRIPDOLJQDQWWUDQVIRUPDWLRQZKLFKFRQWDLQJHQHWLFFKDQJHVDW PXOWLSOHFKURPRVRPDODUPVRUDQHXSORLG\DUHHOLJLEOH_. :KHQZHDSSOLHGVL51$PHGLDWHG

PLK1

NQRFNGRZQGLͿHUHQFHVLQUHVSRQVHZHUH REVHUYHG EHWZHHQ WXPRU DQG SUHPDOLJQDQW FHOOV YHUVXV SULPDU\ FHOOV +RZHYHU ZKHQZHDQDO\]HGWKHVHOHFWHGSDQHORIVPDOOPROHFXOHLQKLELWRUVWKHLUSURÀOHVGLG QRWDOZD\VPLPLFWKDWRIVL51$NQRFNGRZQZKLFKZHFRQVLGHUDVWKHPRVWVSHFLÀF ZD\RIWDUJHWLQKLELWLRQ2QO\WKHLQKLELWRUV%,DQG*6.VKRZHGSURÀOHV

in vitro

 WKDW ZHUH FRPSDUDEOH WR WKH VL51$V HYLGHQFH RI WDUJHW VHOHFWLYLW\ 7KLV VHOHFWLYLW\ZDVFRQÀUPHGE\WKHVSHFLÀFHͿHFWVRQWKHFHOOF\FOHGLVWULEXWLRQRISULPDU\ FHOOVWUHDWHGZLWK*6.RU%,7KH*0DUUHVWRINHUDWLQRF\WHVDIWHUGUXJ WUHDWPHQWZDVVLJQLÀFDQWO\VPDOOHUWKDQWKDWGHPRQVWUDWHGIRUWKHSUHQHRSODVWLFDQG +16&&FHOOV7KHVHGLͿHUHQFHVDUHQRWGHSHQGLQJRQWKHSUROLIHUDWLRQUDWHVRIWKH FHOOVDVWKHSULPDU\FHOOVKDYHDFRPSDUDEOHGRXEOLQJWLPHDVWKH98SUH6&&0 FHOOV7DEOH6/LNHO\LWUHODWHVWRWKHDEURJDWLRQRI*6FHOOF\FOHFRQWUROE\ORVV RISDQGWKHDEVHQFHRIIXQFWLRQDOSVLQFH98SUH6&&0FRQWDLQVDQRQVHQVH

TP53

PXWDWLRQ_. ,WKDVEHHQVKRZQE\RWKHUVWKDW3/.LQKLELWLRQLQSULPDU\FHOOVFDXVHVVHQHVFHQFH YLDDSGHSHQGHQWSDWKZD\ZKLFKPLJKWDSSO\WRWKHSULPDU\FHOOVXVHGLQRXU VWXGLHVDVZHOO7KLVFDQEHPLVVHGZKHQFHOOYLDELOLW\LVH[DPLQHGE\PHWDEROLFDVVD\V DVVHQHVFHQWFHOOVUHPDLQPHWDEROLFDOO\DFWLYH_{+RZHYHUVHQHVFHQFHVHHPVQRWWR} KDYHDQ\FOLQLFDOFRQVHTXHQFHV3KDVH,VWXGLHVRI%,DQG*6.LQSDWLHQWV ZLWK VROLG WXPRUV GHPRQVWUDWHG PRGHUDWH DQWLWXPRU HͿHFWV ZKLOH GRVH OLPLWLQJ WR[LFLWLHVZHUHPDLQO\P\HORVXSSUHVVLRQDQGQRWPXFRVLWLVZKLFKLVDFOLQLFDOVLJQ WKDWFRXOGUHVXOWIURPVHQHVFHQFH_{6LQFHWKHUHDUHQRHVWDEOLVKHGSUHFDQFHUPRXVH}

 

3

ZLWKRUPRUHUHGXFWLRQRIFHOOYLDELOLW\ZHUHFRQVLGHUHGWRUHSUHVHQWYDOLGKLWV 1H[WZHGHÀQHGZKLFKRIWKHUHPDLQLQJJHQHVDUHGUXJJDEOHPDNLQJXVHRISXEOLFO\ DYDLODEOHGDWDEDVHVDQGFKHUU\SLFNLQJZDVSHUIRUPHGEDVHGRQWKHDYDLODELOLW\RI VPDOOPROHFXOHLQKLELWRUV

Deconvolution and serial dilution analysis

0XOWLSOH FHOO OLQHV ZHUH IRUZDUG WUDQVIHFWHG ZLWK WKH WDUJHW VL51$V DQG ZLWK WKH QRQWDUJHWLQJVL&21752/VL&21'KDUPDFRQ*(DQGD60$57SRROGLUHFWHG against

Ubiquitin B (UBB

 'KDUPDFRQ *(_{ DV QHJDWLYH DQG SRVLWLYH FRQWURO}

UHVSHFWLYHO\ )RU GHFRQYROXWLRQ D PLK1 60$57SRRO ZDV XVHG WRJHWKHU ZLWK IRXU VHSDUDWH VL51$V $W  KRXUV SRVWWUDQVIHFWLRQ FHOO YLDELOLW\ ZDV PHDVXUHG DV GHVFULEHGDERYH3HUFHQWDJHVRIFHOOYLDELOLW\ZHUHFDOFXODWHGUHODWLYHWRWKHQHJDWLYH FRQWUROV)RUVORZHUGLYLGLQJFHOOOLQHVVXFKDV98SUH6&&+1DQG98SUH6&& +1 WKH LQFXEDWLRQ SHULRG DIWHU WUDQVIHFWLRQ ZDV DGMXVWHG DFFRUGLQJ WR WKH GRXEOLQJWLPHRIWKHFHOOVWRHQVXUHDWOHDVWWKUHHSRSXODWLRQGRXEOLQJV7DEOH67KH FXOWXUHPHGLXPZDVUHIUHVKHGGXULQJWKHVHORQJHULQFXEDWLRQVWRSUHYHQWVWDUYDWLRQ )RU WKH VL51$ GLOXWLRQ UDQJH WKH WDUJHW 60$57SRRO ZDV GLOXWHG ZLWK [ VL51$ EXͿHU'KDUPDFRQ*(WRREWDLQFRQFHQWUDWLRQVUDQJLQJIURPQPROWLOOQPRO

Expression microarray

&OLQLFDO VSHFLPHQV RI D SDQHO RI  +16&&V DQG FRUUHVSRQGLQJ PDFURVFRSLFDOO\ QRUPDOPXFRVDDGMDFHQWWRWKHWXPRUZHUHFROOHFWHGDVGHVFULEHG_{,QVKRUWPDOHV}

DQGIHPDOHVZHUHLQFOXGHGZLWKDQDYHUDJHDJHRI“\HDUVUDQJH 6SHFLPHQVZHUHREWDLQHGIURPWKHRUDOFDYLW\RXWRIDQGWKHRURSKDU\Q[  RXW RI   DQG DOO PXFRVD VDPSOHV ZHUH MXGJHG IUHH IURP G\VSODVLD $OO RURSKDU\QJHDOWXPRUVZHUH+39QHJDWLYH51$ZDVLVRODWHGZLWK75,]RODQDO\]HG RQDQ$JLOHQW%LRDQDO\]HU$JLOHQWDQGFRPSOHPHQWDU\'1$F'1$V\QWKHVLV ZDVSHUIRUPHG$JLOHQW([SUHVVLRQSURÀOHVZHUHGHWHUPLQHGE\PLFURDUUD\ð. :KROH+XPDQ*HQRPH$UUD\V*)K\EULGL]DWLRQDQGUHDGRXWZDVSHUIRUPHG RQD*%PLFURDUUD\VFDQQHUV\VWHP$JLOHQW'DWDDQDO\VLVZDVSHUIRUPHGDV GHVFULEHG SUHYLRXVO\_{ 'DWD KDYH EHHQ XSORDGHG WR *(2 ZLWK DFFHVVLRQ QXPEHU}

*6(

Quantitative RT-PCR

51$ZDVFROOHFWHGKRXUVDIWHUWUDQVIHFWLRQZLWKWKH51HDV\PLFURNLW4LDJHQ F'1$ ZDV SURGXFHG IURP  QJ 51$ ZLWK D F'1$ UHYHUVH WUDQVFULSWDVH NLW $SSOLHG%LRV\VWHPV)RUTXDQWLWDWLYHPHDVXUHPHQWVRI

PLK1

D[8QLYHUVDO3&5 0DVWHUPL[ $SSOLHG %LRV\VWHPV WRJHWKHU ZLWK D JHQHVSHFLÀF H[SUHVVLRQ DVVD\ +VBPZDVXVHGշ*OXFXURQLGDVH

GUSB

+VBP_ZDVXVHG

 ۚJ $PSKRWHULFLQ % *LEFR DQG  ۚJ *HQWDP\FLQ 6LJPD$OGULFK 98 SUH6&&0ZDVSUHYLRXVO\JHQHUDWHG_{'DSUHQHRSODVWLFFHOOOLQHGHULYHGIURP}

D OHXNRSODNLD OHVLRQ ZDV D JHQHURXV JLIW RI 'U . +XQWHU LQ  8QLYHUVLW\ RI 6KH΀HOG6KH΀HOG8.DQGZDVSUHYLRXVO\GHVFULEHG_{7KHRWKHUSUHQHRSODVWLFFHOO}

OLQHV98SUH6&&98SUH6&&+1DQG98SUH6&&+1ZHUHJHQHUDWHG DQG FKDUDFWHUL]HG DW RXU RZQ ODE 7DEOH 6 DV GHVFULEHG SUHYLRXVO\_{ 'HWDLOHG}

PROHFXODU FKDUDFWHULVWLFV RI WKHVH DQG RWKHU SUHQHRSODVWLF FXOWXUHV DUH SXEOLVKHG HOVHZKHUH_{$OO SUHQHRSODVWLF FHOO OLQHV ZHUH FXOWXUHG LQ .*06)0 986&&}

SUHYLRXVO\GHVFULEHGDV98ZDVHVWDEOLVKHGE\+HUPVHQ

et al

._{806&&$}

ZDVREWDLQHGIURP'U7&DUH\LQ8QLYHUVLW\RI0LFKLJDQ$QQ$UERU0,_

6:ZDVREWDLQHGIURPWKH$PHULFDQ7\SH&XOWXUH&ROOHFWLRQLQ7KHWXPRU FHOO OLQHV ZHUH FXOWXUHG LQ '0(0 FRPSOHPHQWHG ZLWK  IHWDO FDOI VHUXP DQG  PPRO/ /JOXWDPLQH$OO FHOOV ZHUH FXOWXUHG LQ D KXPLGLÀHG DWPRVSKHUH DW ƒ& XQGHU&22DQGXVHGZLWKLQWKUHHPRQWKVDIWHUWKDZLQJ$XWKHQWLFDWLRQRIWKH

FHOO OLQHV ZDV FRQGXFWHG UHJXODUO\ EDVHG RQ PRUSKRORJLFDO FKDUDFWHULVWLFV

TP53

PXWDWLRQVDQGRWKHUJHQHWLFPDUNHUV siRNA re-screen VL51$VZHUHVHOHFWHGIURPSUHYLRXVJHQRPHZLGHVFUHHQVDQGVXEVHTXHQWO\WHVWHG LQDVXEOLEUDU\IRUPDWRQ98SUH6&&0DQGWKHWZRRULJLQDOFHOOOLQHVXVHG98 6&&DQG6:_{7KHVXEOLEUDU\ZDVRUGHUHGDVVL51$60$57SRROVGHULYHG} IURPWKHVL$55$<+XPDQ*HQRPHOLEUDU\'KDUPDFRQ*()URPWKHRULJLQDO VL51$SRROVWKDWZHUHVFRUHGDVKLWEDVHGRQ=VFRUHVUHPDLQHGDVERQDÀGH JHQHVDQGZHUHQRORQJHULQWKHSURGXFWOLVWDVWKHJHQHVZHUHUHWLUHG$OOWKUHH FHOOOLQHVZHUHIRUZDUGWUDQVIHFWHGLQWULSOLFDWHZLWKQPRORIHYHU\60$57SRRO LQFOXGLQJ LQGLYLGXDO SRVLWLYH DQG QHJDWLYH FRQWUROV WRJHWKHU ZLWK 'KDUPD)(&7 'KDUPDFRQ *( )RU 98SUH6&&0 986&& DQG 6:  ۚO  ۚO DQGۚO'KDUPD)(&7SHUZHOOUHVSHFWLYHO\ZDVGHWHUPLQHGDVEHLQJRSWLPDO $IWHUKRXUVRILQFXEDWLRQFHOOYLDELOLW\ZDVGHWHUPLQHGXVLQJWKH&HOO7LWHU%OXH &7% UHDJHQW DVVD\ 3URPHJD DW D  GLOXWLRQ ZLWK PHGLXP 7KH UHDFWLRQ ZDV VWRSSHGDIWHUKRXUVE\DGGLQJVRGLXPGRGHF\OVXOIDWH$SSOLFKHP3DQUHDF )OXRUHVFHQFH ZDV PHDVXUHG ZLWK DQ ,QÀQLWH ) PLFURSODWH UHDGHU 7HFDQ ZLWK H[FLWDWLRQZDYHOHQJWKVHWDWQPDQGHPLVVLRQZDYHOHQJWKDWQP

Screen data analysis

7RLGHQWLI\VL51$VWKDWFDXVHGVLJQLÀFDQWORVVRIFHOOYLDELOLW\UDZPHGLDQYLDELOLW\ YDOXHV ZHUH WUDQVIRUPHG LQWR SHUFHQWDJHV IRU HYHU\ FHOO OLQH DV IROORZV 7R FRUUHFW IRUGLͿHUHQFHVLQWUDQVIHFWLRQH΀FLHQF\EHWZHHQFHOOOLQHVWKHPD[LPXPDFKLHYDEOH UDQJHRIWKHSRVLWLYHFRQWUROVFRPSDUHGWRWKHQHJDWLYHFRQWUROVZDVVHWDW:HOOV

 

3

,PPXQRÁXRUHVFHQFHVWDLQLQJRIPLWRWLFVSLQGOHV 98SUH6&&0FHOOVZHUHJURZQRQZHOO/DE7HNFKDPEHUVOLGHV7KHUPR)LVKHU 6FLHQWLÀFDQGWUHDWHGZLWKۚ0*6.IRUKRXUV7KHFHOOVZHUHÀ[HGDQG VWDLQHGWRYLVXDOL]HWKHPLWRWLFVSLQGOHVDVGHVFULEHG_{5HSUHVHQWDWLYHSLFWXUHVZHUH} VHOHFWHGDQGEDFNJURXQGVWDLQLQJZDVÀOWHUHGXVLQJ,PDJH- HNSCC xenograft study

+16&& FHOO OLQH 806&&$ ZDV VXEFXWDQHRXVO\ LQMHFWHG LQ ERWK ÁDQNV RI ZHHNROGIHPDOHDWK\PLF1XGH)R[Qnu_{PLFH(QYLJRZLWKð}_{FHOOVSHUVLWH}

$SSUR[LPDWHO\ZHHNVODWHUPLFHn ZHUHUDQGRPL]HGLQWRDVROYHQWFRQWURO %,*6.DQGDFKHPRUDGLDWLRQJURXS573ODW7KHODWWHUFRQFRPLWDQWO\ WUHDWHGZLWKUDGLRWKHUDS\*\DQGFLVSODWLQPJNJDIUHTXHQWO\XVHGWUHDWPHQW VWUDWHJ\ LQ FOLQLFDO FDUH DOEHLW ZLWK D PXFK KLJKHU WRWDO LUUDGLDWLRQ GRVH %RWK LQKLELWRUVZHUHIRUPXODWHGLQ'062DQGVROXWROZKLFKZDVXVHGDVWKH VROYHQWJLYHQWRWKHFRQWUROPLFH0LFHZHUHWUHDWHGDFFRUGLQJWRWKHVFKHPHGHSLFWHG LQ7DEOH67XPRUVZHUHPHDVXUHGHYHU\RWKHUGD\E\FDOLSHUVDQGWXPRUYROXPH ZDVTXDQWLÀHGZLWKWKHIRUPXODOHQJWK[ZLGWK[GHSWK[$QLPDOZHLJKWZDV GHWHUPLQHGDVLQGLFDWRURIWUHDWPHQWWR[LFLW\$OODQLPDOH[SHULPHQWVZHUHSHUIRUPHG DFFRUGLQJWR'XWFKDQG(8OHJLVODWLRQVDQGWKHSURWRFROZDVDSSURYHGE\WKH ,QVWLWXWLRQDO5HYLHZ%RDUGRQDQLPDOH[SHULPHQWDWLRQ Statistical analysis

'LͿHUHQFHV EHWZHHQ JURXSV ZHUH DVVHVVHG E\ WDLOHG 6WXGHQW

t

 WHVW RU 0DQQ :KLWQH\8WHVW'LͿHUHQFHVLQWUHDWPHQWHͿHFWRQFHOOF\FOHGLVWULEXWLRQZHUHDVVHVVHG E\DJHQHUDOL]HGOLQHDUPRGHOZLWKD3RLVVRQHUURUDQGDORJDULWKPLFOLQN'LͿHUHQFHV LQVSLQGOHDVVHPEO\ZHUHDVVHVVHGE\WKH)LVKHUH[DFWWHVW$QDO\VLVRIVXUYLYDORIWKH WUHDWHGPLFHZDVSHUIRUPHGE\WKH.DSODQ0HLHUPHWKRGDQGGLͿHUHQFHVEHWZHHQ WKHJURXSVZDVDVVHVVHGE\WKH/RJUDQNWHVW0DQWHO&R[$

P

YDOXHOHVVWKDQ ZDVFRQVLGHUHGVLJQLÀFDQW ACKNOWLEDGEMENTS 7KHDXWKRUVWKDQN,GDYDQGHU0HXOHQ0XLOHPDQIRUKHUFRQWULEXWLRQWRWKHVL51$ VFUHHQVDQG5HQpHGH0HQH]HVIRUWKHVWDWLVWLFDODQDO\VLVRIRXUÁRZF\WRPHWU\GDWD DVDQLQWHUQDOUHIHUHQFH5HDFWLRQVZHUHSHUIRUPHGRQWKH$%,3ULVPVHTXHQFH

GHWHFWRU V\VWHP 7DTPDQ3&5 $SSOLHG %LRV\VWHPV ([SUHVVLRQ RI

PLK1

 ZDV FDOFXODWHGUHODWLYHWR

GUSB

ӹ&WPHWKRG

Western blot

&HOOV ZHUH O\VHG LQ 5,3$ EXͿHU 7KHUPR 6FLHQWLÀF VXSSOHPHQWHG ZLWK D +$/7 SURWHDVH DQG SKRVSKDWDVH LQKLELWRU FRFNWDLO 7KHUPR 6FLHQWLÀF  KRXUV DIWHU WUDQVIHFWLRQZLWKVL51$V/\VDWHVZHUHORDGHGRQWR0LQL3URWHDQ7*;JHOV %LR5DGDQGWUDQVIHUUHGWRD39')PHPEUDQH0HUFN0LOOLSRUH:HVWHUQEORWZDV SHUIRUPHG ZLWK UDEELW DQWL3/.  &HOO 6LJQDOLQJ DQG PRXVH DQWLշDFWLQ FORQH$&6LJPD$OGULFKDQWLERGLHV3URWHLQVZHUHYLVXDOL]HGZLWKWKH ÁXRUHVFHQWO\ODEHOHGDQWLERGLHVJRDWDQWLUDEELW,5'\H&:DQGJRDWDQWLPRXVH ,5'\H 5'  /,&25 %LRVFLHQFHV %ORWV ZHUH VFDQQHG RQ WKH 2G\VVH\ LQIUDUHGLPDJLQJV\VWHP/,&25%LRVFLHQFHV3URWHLQOHYHOVZHUHVWDQGDUGL]HGWR WKHշDFWLQOHYHOVDQGTXDQWLÀFDWLRQZDVSHUIRUPHGZLWK,PDJH-6RIWZDUH1,+

Dose response curves

&HOOV ZHUH WUHDWHG ZLWK IRXU VPDOO PROHFXOH LQKLELWRUV GLUHFWHG DJDLQVW 3/. *6. $[RQ 0HGFKHP %, $[RQ 0HGFKHP 21 6HOOHFNFKHP DQG +01 6HOOHFNFKHP DOO GLVVROYHG LQ '062 &HOOV ZHUH WUHDWHG ZLWK FRQFHQWUDWLRQVUDQJLQJIURPWLOOۚ0ZKHUHFRQFHQWUDWLRQVRI'062ZHUHDOZD\V EHORZ$IWHUKRXUVRILQFXEDWLRQWKHHͿHFWVRQFHOOYLDELOLW\ZHUHPHDVXUHG DVGHVFULEHGDERYH

Cell-cycle analysis

&HOOVZHUHWUHDWHGIRUKRXUVZLWKHLWKHUۚ0%,RUۚ0*6. RU ZLWK  QPRO RI WKH VL51$ 60$57SRRO RU VL&21 1H[W FHOOV ZHUH À[HG ZLWK LFHFROG(W2+LQ3%6/RQ]D%HIRUHVWDLQLQJFHOOVZHUHLQFXEDWHGZLWKۚO 51$VHۚJPO6LJPD$OGULFKIRUPLQXWHVDWURRPWHPSHUDWXUH7KHQ ۚO 3URSLGLXP ,RGLGH 3,  ۚJPO 6LJPD$OGULFK ZDV DGGHG DQG '1$ FRQWHQW PHDVXUHPHQWV ZHUH SHUIRUPHG RQ D )$&6 %' /65 ,, )RUWHVVD %' %LRVFLHQFHV &HOO F\FOH GLVWULEXWLRQ ZDV DQDO\]HG ZLWK %' )$&6 ',9$ VRIWZDUH 9 %' %LRVFLHQFHV

7KHGDWDZDVDQDO\]HGXVLQJDJHQHUDOL]HGOLQHDUPRGHOZLWKD3RLVVRQHUURUDQGD ORJDULWKPLFOLQN7KHOLQHDUSUHGLFWLRQLQFOXGHGFHOOOLQHDQGWUHDWPHQWHͿHFWVDVZHOO DVDQLQWHUDFWLRQEHWZHHQWKHVHWZR

 

3

 7\QHU-:'HLQLQJHU0:/RULDX[00&KDQJ%+*RWOLE-5:LOOLV6*et al. RNAi screen for rapid

WKHUDSHXWLFWDUJHWLGHQWLÀFDWLRQLQOHXNHPLDSDWLHQWVProc Natl Acad Sci U S A106²  0DUWHQVGH .HPS 65 1DJHO 5 6WLJWHUYDQ :DOVXP 0 YDQ GHU 0HXOHQ ,+ YDQ %HXVHFKHP 9:

%UDDNKXLV%-0et al. )XQFWLRQDOJHQHWLFVFUHHQVLGHQWLI\JHQHVHVVHQWLDOIRUWXPRUFHOOVXUYLYDOLQKHDG DQGQHFNDQGOXQJFDQFHUClin Cancer Res19²

 1DJHO56WLJWHUYDQ:DOVXP0%XLM]H0YDQGHQ%HUJ-YDQGHU0HXOHQ,++RG]LF-et al. *HQRPH ZLGHVL51$6FUHHQ,GHQWLÀHVWKH5DGLRVHQVLWL]LQJ(ͿHFWRI'RZQUHJXODWLRQRI0$67/DQG)2;0LQ NSCLC. Mol Cancer Ther14²

 =LWRXQL61DEDLV&-DQD6&*XHUUHUR$%HWWHQFRXUW'LDV03ROROLNHNLQDVHVVWUXFWXUDOYDULDWLRQV OHDGWRPXOWLSOHIXQFWLRQVNat Rev Mol Cell Biol15²

 1RJDZD0<XDVD7.LPXUD67DQDND0.XURGD-6DWR.et al.,QWUDYHVLFDODGPLQLVWUDWLRQRIVPDOO LQWHUIHULQJ51$WDUJHWLQJ3/.VXFFHVVIXOO\SUHYHQWVWKHJURZWKRIEODGGHUFDQFHU-&OLQ,QYHVW

115²

 5HQQHU$* 'RV 6DQWRV 6 5HFKHU & %DLOO\ & &UHDQFLHU / .UXF]\QVNL$ et al. 3ROROLNH NLQDVH  LV RYHUH[SUHVVHGLQDFXWHP\HORLGOHXNHPLDDQGLWVLQKLELWLRQSUHIHUHQWLDOO\WDUJHWVWKHSUROLIHUDWLRQRI OHXNHPLFFHOOVBlood114²

 6WUHEKDUGW.8OOULFK$7DUJHWLQJSROROLNHNLQDVHIRUFDQFHUWKHUDS\Nat Rev Cancer6²  GH%RHU'9%ULQN$%XLM]H06WLJWHUYDQ:DOVXP0+XQWHU.'<OVWUD%et al. (VWDEOLVKPHQWDQG

*HQHWLF/DQGVFDSHRI3UHFDQFHU&HOO0RGHO6\VWHPVIURPWKH+HDGDQG1HFN0XFRVDO/LQLQJMol Cancer Res17²

25 Sch|ͿVNL33ROROLNHNLQDVH3/.LQKLELWRUVLQSUHFOLQLFDODQGHDUO\FOLQLFDOGHYHORSPHQWLQRQFRORJ\ Oncologist14²

 &KRL 0 .LP : &KHRQ 0* /HH &: .LP -( 3ROROLNH NLQDVH  LQKLELWRU %, FDXVHV PLWRWLF FDWDVWURSKHIROORZLQJDFWLYDWLRQRIWKHVSLQGOHDVVHPEO\FKHFNSRLQWLQQRQVPDOOFHOOOXQJFDQFHUFHOOV Cancer Lett357²  6XPDUD,*LPpQH]$ELáQ-)*HUOLFK'+LURWD7.UDIW&GHOD7RUUH&et al.5ROHVRISROROLNHNLQDVH LQWKHDVVHPEO\RIIXQFWLRQDOPLWRWLFVSLQGOHVCurr Biol14²  +ROPVWUXS39HGWRIWH35HLEHO-6WROW]H./RQJWHUPWUHDWPHQWRXWFRPHRIRUDOSUHPDOLJQDQWOHVLRQV Oral Oncol42²  .QHFKW5(OH]52HFKOHU06ROEDFK&YRQ,OEHUJ&6WUHEKDUGW.3URJQRVWLFVLJQLÀFDQFHRISROR OLNHNLQDVH3/.H[SUHVVLRQLQVTXDPRXVFHOOFDUFLQRPDVRIWKHKHDGDQGQHFNCancer Res59 ²  YDQGH:HHUGW%&00HGHPD5+3ROROLNHNLQDVHVDWHDPLQFRQWURORIWKHGLYLVLRQ&HOO&\FOH 5²

 1DSLHU 66 6SHLJKW 30 1DWXUDO KLVWRU\ RI SRWHQWLDOO\ PDOLJQDQW RUDO OHVLRQV DQG FRQGLWLRQV DQ RYHUYLHZRIWKHOLWHUDWXUH-2UDO3DWKRO0HG37²

 %UHPPHU -) %UDNHQKRͿ 5+ %URHFNDHUW 0$ %HOLHQ -$ /HHPDQV &5 %ORHPHQD ( et al. Prognostic YDOXHRI'1$SORLG\VWDWXVLQSDWLHQWVZLWKRUDOOHXNRSODNLDOral Oncol47²

 =KDQJ/3RK&):LOOLDPV0/DURQGH'0%HUHDQ.*DUGQHU3-et al./RVVRIKHWHUR]\JRVLW\/2+ SURÀOHVYDOLGDWHGULVNSUHGLFWRUVIRUSURJUHVVLRQWRRUDOFDQFHUCancer Prev Res (Phila)5²   .LP+-&KR-+.LP-5'RZQUHJXODWLRQRI3ROROLNHNLQDVHLQGXFHVFHOOXODUVHQHVFHQFHLQKXPDQ SULPDU\FHOOVWKURXJKDSGHSHQGHQWSDWKZD\-*HURQWRO$%LRO6FL0HG6FL68²  5RQLQVRQ,%7XPRUFHOOVHQHVFHQFHLQFDQFHUWUHDWPHQWCancer Res63² REFERENCES  )HUOD\-6KLQ+5%UD\))RUPDQ'0DWKHUV&3DUNLQ'0(VWLPDWHVRIZRUOGZLGHEXUGHQRIFDQFHU LQ*/2%2&$1,QW-&DQFHU127²  /HHPDQV&5%UDDNKXLV%-0%UDNHQKRͿ5+7KHPROHFXODUELRORJ\RIKHDGDQGQHFNFDQFHUNat Rev Cancer11²  YDQGHU:DDO,3RWHQWLDOO\PDOLJQDQWGLVRUGHUVRIWKHRUDODQGRURSKDU\QJHDOPXFRVDWHUPLQRORJ\ FODVVLÀFDWLRQDQGSUHVHQWFRQFHSWVRIPDQDJHPHQWOral Oncol45²

 %UDDNKXLV %-0 7DERU 03 .XPPHU -$ /HHPDQV &5 %UDNHQKRͿ 5+ $ JHQHWLF H[SODQDWLRQ RI 6ODXJKWHU·V FRQFHSW RI ÀHOG FDQFHUL]DWLRQ HYLGHQFH DQG FOLQLFDO LPSOLFDWLRQV Cancer Res  63 ²

 7DERU03%UDNHQKRͿ5+YDQ+RXWHQ900.XPPHU-$6QHO0+-6QLMGHUV3-)et al. Persistence

RIJHQHWLFDOO\DOWHUHGÀHOGVLQKHDGDQGQHFNFDQFHUSDWLHQWV%LRORJLFDODQGFOLQLFDOLPSOLFDWLRQVClin Cancer Res7²

 7DERU03%UDDNKXLV%-YDQGHU:DO-(YDQ'LHVW3-/HHPDQV&5%UDNHQKRͿ5+et al. Comparative PROHFXODU DQG KLVWRORJLFDO JUDGLQJ RI HSLWKHOLDO G\VSODVLD RI WKH RUDO FDYLW\ DQG WKH RURSKDU\Q[ - Pathol199²

 3RK&)=KDQJ/$QGHUVRQ':'XUKDP-6:LOOLDPV303ULGG\5:et al.)OXRUHVFHQFHYLVXDOL]DWLRQ GHWHFWLRQRIÀHOGDOWHUDWLRQVLQWXPRUPDUJLQVRIRUDOFDQFHUSDWLHQWVClin Cancer Res12² 

 *UDYHODQG$3*ROXVLQVNL3-%XLM]H0'RXPD56RQV1.XLN'-et al./RVVRIKHWHUR]\JRVLW\DWS DQGSLPPXQRSRVLWLYLW\LQVXUJLFDOPDUJLQVSUHGLFWORFDOUHODSVHLQKHDGDQGQHFNVTXDPRXVFHOO carcinoma. ,QW-&DQFHU128²

 7DERU03%UDNHQKRͿ5+5XLMWHU6FKLSSHUV+-.XPPHU-$/HHPDQV&5%UDDNKXLV%-*HQHWLFDOO\ DOWHUHGÀHOGVDVRULJLQRIORFDOO\UHFXUUHQWKHDGDQGQHFNFDQFHUDUHWURVSHFWLYHVWXG\Clin Cancer Res 10²

 6FKDDLM9LVVHU7%*UDYHODQG$3*DXFL6%UDDNKXLV%-%XLM]H0+HFN$-et al. 'LͿHUHQWLDO3URWHRPLFV ,GHQWLÀHV3URWHLQ%LRPDUNHUV7KDW3UHGLFW/RFDO5HODSVHRI+HDGDQG1HFN6TXDPRXV&HOO&DUFLQRPDV Clin Cancer Res15²

 5RHVFK(O\ 0 1HHV 0 .DUVDL 6 5XHVV$ %RJXPLO 5 :DUQNHQ 8 et al. 3URWHRPLF DQDO\VLV UHYHDOV VXFFHVVLYHDEHUUDWLRQVLQSURWHLQH[SUHVVLRQIURPKHDOWK\PXFRVDWRLQYDVLYHKHDGDQGQHFNFDQFHU Oncogene26²  3RK&)$QGHUVRQ':'XUKDP-6&KHQ-%HUHDQ.:0DF$XOD\&(et al.)OXRUHVFHQFH9LVXDOL]DWLRQ *XLGHG6XUJHU\IRU(DUO\6WDJH2UDO&DQFHU-$0$2WRODU\QJRO+HDG1HFN6XUJ142²  YDQ=HHEXUJ+-7*UDYHODQG$3%ULQN$1JX\HQ0/HHPDQV&5%ORHPHQD(et al.*HQHUDWLRQRI SUHFXUVRUFHOOOLQHVIURPSUHQHRSODVWLFÀHOGVVXUURXQGLQJKHDGDQGQHFNFDQFHUVHead Neck35 ²

 0F*UHJRU ) :DJQHU ( )HOL[ ' 6RXWDU ' 3DUNLQVRQ . +DUULVRQ 3 ,QDSSURSULDWH 5HWLQRLF $FLG 5HFHSWRUD([SUHVVLRQLQ2UDO'\VSODVLDV&RUUHODWLRQZLWK$FTXLVLWLRQRIWKH,PPRUWDO3KHQRW\SH Cancer Res57²

 0F*UHJRU ) 0XQWRQL $ )OHPLQJ - %URZQ - )HOL[ '+ 0DF'RQDOG '* et al. 0ROHFXODU FKDQJHV DVVRFLDWHGZLWKRUDOG\VSODVLDSURJUHVVLRQDQGDFTXLVLWLRQRILPPRUWDOLW\SRWHQWLDOIRULWVUHYHUVDOE\ D]DF\WLGLQHCancer Res62²

 /DZUHQFH06RXJQH]&/LFKWHQVWHLQ/&LEXOVNLV./DQGHU(*DEULHO6et al. Comprehensive genomic FKDUDFWHUL]DWLRQRIKHDGDQGQHFNVTXDPRXVFHOOFDUFLQRPDVNature517²

 

3

SUPPLEMENTARY DATA Genome-wide siRNA screen 319 lethal siRNA pools 98 lethal siRNA pools Selection of druggable targets Polo-like kinase 1 2 tumor cell lines

VU-preSCC-M3

Primary cells and other preneoplastic cell lines

Figure S1: Screening of VU-preSCC-M3 with a panel of 319 ‘tumor-lethal’ siRNAs. 2XWOLQHRIWKH

VL51$VFUHHQLQJDQGFDQGLGDWHWDUJHWLGHQWLÀFDWLRQSURFHGXUH

 2OPRV'%DUNHU'6KDUPD5%UXQHWWR$7<DS7$7DHJWPH\HU$%et al.3KDVH,VWXG\RI*6. DVSHFLÀFDQGFRPSHWLWLYH3ROROLNHNLQDVHLQKLELWRULQSDWLHQWVZLWKDGYDQFHGVROLGPDOLJQDQFLHV Clin Cancer Res17²

 6FK|ͿVNL3$ZDGD$'XPH]+*LO7%DUWKRORPHXV6:ROWHU3et al.$SKDVH,GRVHHVFDODWLRQVWXG\ RIWKHQRYHO3ROROLNHNLQDVHLQKLELWRUYRODVHUWLE%,LQSDWLHQWVZLWKDGYDQFHGVROLGWXPRXUV(XU -&DQFHU48²

 5XGROSK'6WHHJPDLHU0+RͿPDQQ0*UDXHUW0%DXP$4XDQW-et al.%,D3ROROLNHNLQDVH LQKLELWRUZLWKLPSURYHGSKDUPDFRNLQHWLFSURÀOHDQGEURDGDQWLWXPRUDFWLYLW\ Clin Cancer Res15 ²

 +HUPVHQ0$-$-RHQMH+$UZHUW):HOWHUV0-3%UDDNKXLV%-0%DJQD\0et al.&HQWURPHULFEUHDNDJH DV D PDMRU FDXVH RI F\WRJHQHWLF DEQRUPDOLWLHV LQ RUDO VTXDPRXV FHOO FDUFLQRPD Genes Chromosomes Cancer15²

 /LQ&-*UDQGLV-5&DUH\7(*ROOLQ60:KLWHVLGH7/.RFK:0et al.+HDGDQGQHFNVTXDPRXVFHOO FDUFLQRPDFHOOOLQHVHVWDEOLVKHGPRGHOVDQGUDWLRQDOHIRUVHOHFWLRQHead Neck29²  7LHGHPDQQ 5( =KX <; 6FKPLGW - 6KL &; 6HUHGXN & <LQ + et al. ,GHQWLÀFDWLRQ RI PROHFXODU

YXOQHUDELOLWLHV LQ KXPDQ PXOWLSOH P\HORPD FHOOV E\ 51$ LQWHUIHUHQFH OHWKDOLW\ VFUHHQLQJ RI WKH GUXJJDEOHJHQRPHCancer Res72²

 0DUWHQVGH.HPS65%ULQN$YDQGHU0HXOHQ,+GH0HQH]HV5;WH%HHVW'(/HHPDQV&5et al. The )$%5&$3DWKZD\,GHQWLÀHGDVWKH0DMRU3UHGLFWRURI&LVSODWLQ5HVSRQVHLQ+HDGDQG1HFN&DQFHU E\)XQFWLRQDO*HQRPLFVMol Cancer Ther16

 0DUWHQVGH.HPS65%ULQN$6WLJWHUYDQ:DOVXP0'DPHQ-0$5XVWHQEXUJ):X7et al. &' PDUNVDVXESRSXODWLRQRIKHDGDQGQHFNVTXDPRXVFHOOFDUFLQRPDFHOOVZLWKVWHPFHOOSURSHUWLHVStem Cell Res10²

 $HUWV -/ *RQ]DOHV 0, 7RSDOLDQ 6/ 6HOHFWLRQ RI DSSURSULDWH FRQWURO JHQHV WR DVVHVV H[SUHVVLRQ RI WXPRUDQWLJHQVXVLQJUHDOWLPH573&5Biotechniques36²

 

3

Figure S3: Microarray gene expression data revealed elevated levels of PLK1 in tumor samples.

'DWDGHULYHGIURPSDLUVRIWXPRUDQGQRUPDOPXFRVDWLVVXHUHYHDOHGDIROGXSUHJXODWLRQRI

PLK1H[SUHVVLRQRQDYHUDJHLQWKHWXPRUVDPSOHV'DWDLVSUHVHQWHGDVER[SORWVDQGWKHWKLFNOLQHV

UHSUHVHQWWKHPHGLDQYDOXHV

Figure S2: Cluster analysis based on the 98 essential genes of preneoplastic cell line VU-preSCC-M3. 0XOWLSOHSDWKZD\VDQGFHOOXODUIXQFWLRQVZHUHIRXQGWREHVLJQLÀFDQWO\RYHUUHSUHVHQWHGLQWKLV

JHQHVHWZKLFKLQFOXGHVPLWRVLVFHOOF\FOHUHJXODWLRQDQGULERVRPDOSURFHVVLQJ3ROROLNHNLQDVH DQLPSRUWDQWFHOOF\FOHUHJXODWRUDQGDGUXJJDEOHNLQDVHLVHQFLUFOHG7KHOLQHVUHSUHVHQWWKHFRQÀ GHQFHOHYHOVRIWKHQHWZRUNVWURQJHUDVVRFLDWLRQVDUHUHSUHVHQWHGE\WKLFNHUOLQHV675,1*GDWDEDVH YHUVLRQ

 

3

PI Intensity 0.25 μM BI6727 PI Intensity 0.07 μM GSK461364 PI Intensity Untreated )LJXUH6&HOOF\FOHSURÀOHVRI98SUH6&&0VKRZD*0DUUHVWXSRQ3/.LQKLELWLRQ KRXUWUHDWPHQWZLWKHLWKHU*6.RU%,UHVXOWHGLQDFOHDUDFFXPXODWLRQRIWKHSUHQHRSODVWLF FHOOVLQWKH*0SKDVH6KRZQKHUHDUHUHSUHVHQWDWLYHÀJXUHVRIRQHH[SHULPHQWZKHUH3,LQWHQVLW\ represents the DNA content.

Figure S4: PLK1 protein levels in several cell models and after siRNA transfection. a. Correspon

ding to the increased PLK1H[SUHVVLRQLQWXPRUPDWHULDOZHDOVRIRXQGHOHYDWHGOHYHOVRI3/.SUR WHLQ H[SUHVVLRQ LQ RXU SUHFDQFHU FHOO PRGHOV FRPSDUHG WR SULPDU\ NHUDWLQRF\WHV %DUV UHSUHVHQW 3/. H[SUHVVLRQ OHYHOV UHODWLYH WR WKH շDFWLQ OHYHOV XVHG DV ORDGLQJ FRQWURO b.$QDO\VLV RI 3/. SURWHLQ OHYHOV LQ 806&&$ UHYHDOHG WKDW WUDQVIHFWLRQ ZLWK WKH PLK1 60$57SRRO DQG WKH IRXU LQGLYLGXDOVL51$VUHVXOWHGLQDWOHDVWGHFUHDVHRIWKHSURWHLQOHYHOV%DUVUHSUHVHQWSURWHLQH[ SUHVVLRQUHODWLYHO\WRWKH3/.SURWHLQOHYHORIWKHVL&21752/WUDQVIHFWHGFHOOV

 

3

VU-preSCC-M3 Cell viability (%) SW1573 Cell viability (%) VU-SCC-120 Cell viability (%)

CKAP5 38.1 CKAP5 10.0 CKAP5 13.6

&2/$  &2/$  &2/$ 

COL20A1 24.7 COL20A1 16.4 COL20A1 8.2

COPA 15.6 COPA -10.7 COPA -1.4

COPB1 22.9 COPB1 -0.2 COPB1 7.7

COPB2 26.8 COPB2 -9.0 COPB2 0.6

&23*  COPG1 30.3 COPG1 2.1

COPZ1 23.8 COPZ1 3.2 COPZ1 17.2

&23=  COPZ2 49.3 &23= 

CRNKL1 40.2 CRNKL1 39.6 &51./ 

CSDC2  CSDC2  CSDC2 

CSNK2A2  CSNK2A2  CSNK2A2 37.5

CSTA  CSTA  CSTA 55.5

&:&  &:&  &:& 

&:&  CWC22 4.3 CWC22 34.0

&;RUI  &;RUI  &;RUI 

''% 52.5 ''%  ''% 

DDX19A 48.3 DDX19A 20.5 DDX19A 23.3

DDX19B 30.7 '';%  DDX19B 23.6

'';;  '';;  '';; 

'';  '';  DDX42 18.0

DHX8 32.9 DHX8 30.9 '+; 

'..  '..  DKK3 41.2

DSE  DSE  DSE 29.5

EFTUD2  EFTUD2  EFTUD2 38.4

(,)$;  (,)$;  (,)$; 

(,)%  (,)%  (,)% 

(,)6  (,)6  EIF2S1 46.6

EIF2S2  EIF2S2  EIF2S2 

(,)6  (,)6  (,)6 

EIF3A 42.3 EIF3A 6.6 EIF3A 41.1

EIF3B 23.9 EIF3B 13.4 EIF3B 47.2

EIF3C 33.0 EIF3C 1.7 EIF3C 7.4

(,)*  EIF3G 7.8 EIF3G 20.8

(,),  EIF3I 5.3 (,), 

EIF4A3 17.6 EIF4A3 23.7 EIF4A3 12.3

(,)(  (,)(  (,)( 

EPRS  EPRS  EPRS 

(36/  (36/  EPS8L1 32.4

(7)  (7)  (7) 

FAU 26.9 FAU 16.2 FAU 17.5

FBXO30 42.5 FBXO30 43.9 )%;2 

7DEOH62YHUYLHZRIWKHVL51$UHVFUHHQSHUIRUPHGRQWKUHHGL;HUHQWFHOOOLQHV

VU-preSCC-M3 Cell viability (%) SW1573 Cell viability (%) VU-SCC-120 Cell viability (%)

$'$076  $'$076  $'$076 

$*)*  AGFG2 33.5 AGFG2 37.5

AHSP 28.8 AHSP 23.3 AHSP 

$/'+$  $/'+$  ALDH18A1 46.3

$1$3&  $1$3&  $1$3& 

$1$3&  ANAPC4 34.4 $1$3& 

$36  $36  AP5S1 44.4

$3&''/  $3&''/  APCDD1L 41.0

AQR  AQR 29.7 AQR 32.5

ARCN1 31.3 ARCN1 -13.6 ARCN1 1.9

$5)*()  $5)*()  $5)*() 

$5+*$3  $5+*$3  ARHGAP20 44.4

$5/  $5/  $5/ 

ARSG 46.9 $56*  ARSG 41.1

ASB16 39.2 $6%  ASB16 43.3

ASNS  ASNS 31.1 ASNS 

$73$3  $73$3  ATP6AP1 17.0

BIRC5  BIRC5  BIRC5 

&RUI  &RUI  &RUI 

&RUI  &RUI  C19orf59 33.4

&RUI  C22orf29 30.7 &RUI 

C8orf31 34.1 C8orf31 5.8 C8orf31 26.5

CACTIN  CACTIN  CACTIN 

CALCOCO2  CALCOCO2  CALCOCO2 

CCBL2  CCBL2  CCBL2 34.5

&&'&  &&'&  &&'& 

CCDC99 40.9 CCDC99 34.4 CCDC99 13.7 &&7  &&7  CCT4 49.6 &&7  &&7  CCT7 45.8 &&7  &&7  CCT8 39.3 &'  &'  CD207 28.0 &'&  &'&  CDC40 45.5 CDC5L  CDC5L  CDC5L 

&'&$  &'&$  CDCA8 34.6

&'.  &'.  &'. 

&'.$  &'.$  CDK11A 35.3

CELF5  CELF5  CELF5 29.8

CENPE  CENPE 21.7 CENPE 18.3

&(3  CEP72 17.6 &(3 

 

3

VU-preSCC-M3 Cell viability (%) SW1573 Cell viability (%) VU-SCC-120 Cell viability (%)

NHP2L1 19.3 NHP2L1 -10.4 NHP2L1 25.5

NSA2  NSA2  NSA2 22.3

NXF1 22.6 NXF1 39.5 NXF1 10.6 2*'+  OGDH  2*'+  25-  25-  25-  3$%31  PABPN1 40.5 3$%31  3&'+%  3&'+%  PCDHB13 36.6 PCF11 22.8 PCF11 35.6 PCF11 22.2

PEPD  PEPD  PEPD 

3(6  PES1 49.0 3(6 

PHF5A  PHF5A  PHF5A 

PLK1 0.5 PLK1 0.8 PLK1 4.9

3/5*  PLRG1 37.6 3/5* 

32/'  32/'  POLD4 32.8

POLR2A 3.8 POLR2A -18.4 POLR2A -3.4

POLR2B  POLR2B 11.8 POLR2B 36.7

POLR2C 32.6 POLR2C 30.0 POLR2C 48.9

POLR2D  POLR2D  POLR2D 14.6

POLR2E  POLR2E 6.0 POLR2E 26.6

POLR2F 49.3 POLR2F 10.8 POLR2F 16.5

32/5*  POLR2G 45.9 POLR2G 14.0

POLR2I  POLR2I  POLR2I 43.0

32/5(  32/5(  32/5( 

32/5+  POLR3H 36.9 POLR3H 48.5

POMP  POMP 24.4 POMP 35.6

PPAP2C  PPAP2C 40.8 PPAP2C 

35.$*  35.$*  35.$*  PRLH  PRLH  PRLH 45.5 353)  353)  353)  353)  353)  353)  353)$  PRPF38A 16.7 PRPF38A 47.0 353)%  353)%  PRPF4B 37.1 353)  PRPF8 31.4 PRPF8 43.3 360$  PSMA1 25.1 PSMA1 42.5

PSMA2  PSMA2 20.0 PSMA2 28.3

PSMA4 42.3 PSMA4 19.2 360$ 

PSMA5 36.4 PSMA5 40.8 PSMA5 

360$  PSMA6 27.9 PSMA6 48.8

360$  PSMA7 39.9 360$ 

360%  PSMB3 17.7 360% 

PSMB5  PSMB5 25.5 PSMB5 

360%  PSMB6 18.2 360% 

VU-preSCC-M3 Cell viability (%) SW1573 Cell viability (%) VU-SCC-120 Cell viability (%)

*$%5%  *$%5%  *$%5% 

*$%55  *$%55  *$%55 

*13'$  GNPDA1 41.6 *13'$ 

*3;  GPX4 31.5 *3; 

*637  *637  *637 

HAPLN2  HAPLN2 45.9 HAPLN2 

+(6  +(6  HES4 24.8

HNRNPC  HNRNPC 21.5 HNRNPC 

HNRNPK 30.2 HNRNPK 6.0 HNRNPK 0.2

HNRNPL  HNRNPL  HNRNPL 

HNRNPU  HNRNPU  HNRNPU 

IK 25.8 IK 40.9 IK 38.1

INCENP  INCENP  INCENP 49.9

,176  ,176  ,176 

ISCU  ISCU 40.8 ISCU 

,7*$9  ,7*$9  ,7*$9  JAKMIP3 46.9 -$.0,3  -$.0,3  KDM4A 42.6 KDM4A 44.8 .'0$  KIF11 18.1 .,)  KIF11 32.4 KIF18A 32.0 .,)$  KIF18A 17.9 .,)  .,)  .,)  ./+/  ./+/  KLHL1 31.8 KPNB1 47.4 KPNB1 41.5 KPNB1 24.3 KRTAP13-2 48.4 .57$3  KRTAP13-2 36.0

LARS  LARS  LARS 

LCE3A 48.9 LCE3A 16.6 /&($ 

LIF  LIF  LIF 

/2&  /2&  LOC342293 40.8

/2;/  /2;/  /2;/  LPAR3 45.1 /3$5  LPAR3 30.4 /55&  LRRC61 36.9 LRRC61 34.1 /60  /60  /60  0&5  MC3R 14.9 0&5  0('  0('  MED21 34.0 0)$3  MFAP1 29.3 MFAP1 49.8 06$  06$  06$  0675  0675  0675  1$07  1$07  1$07  1$$  1$$  1$$  1$%  1$%  NAB1 47.7

NAPA 45.0 NAPA -11.4 NAPA 14.8

NDE1 42.5 NDE1 24.6 NDE1 15.8

Table S1 (continued) Table S1 (continued)