1509
Vog M, e al. Gu 2024;73:1509–1528. doi:10.1136/gu jnl-2023-331519
Panc eas
O iginal esea ch
Ta ge ing MYC e ec o unc ions in panc ea ic cance
by inhibi ing he ATPaseRUVBL1/2
Ma kus Vog ,1,2 Ne enka Dud a ski S anko ic ,1,2 Yiliam C uz Ga cia,1,2
Julia Ho s e e ,1 Ka ha ina Schneide ,1,2,3 Filiz Kuybu,3 The esa Hauck,3
Bikash Adhika i,1,2 An on Hamann,4 Yamila Rocca,5 La a G ysczyk,1 Benedik Ma in,1
Anneli Gebha d - Wol ,6 A min Wiege ing ,6,7 Ma kus Die enbache ,8,9
Geo g Gas eige ,5 S e an Knapp,4 Die e Sau ,10 Ma in Eile s,6
Ma hias Rosen eld ,11 Flo ian E ha d,12 Seychelle M Vos,3 Elma Wol 1,2
To ci e: Vog M, Dud a ski
S anko icN, C uz Ga ciaY,
e al. Gu
2024;73:1509–1528.
►Addi ional supplemen al
ma e ial is published online
only. To iew, please isi he
jou nal online (h ps:// doi. o g/
10. 1136/ gu jnl- 2023- 331519).
Fo numbe ed a ilia ions see
end o a icle.
Co espondence o
P o esso Elma Wol , Ins i u e o
Biochemis y, Uni e si y o Kiel,
Kiel, Ge many;
elma . wol @ biochem. uni- kiel. de
MV and NDS a e join i s
au ho s.
Recei ed 5 Decembe 2023
Accep ed 15 May 2024
Published Online Fi s
31May2024
© Au ho (s) (o hei
employe (s)) 2024. Re- use
pe mi ed unde CC BY.
Published by BMJ.
ABSTRACT
Objec i e The hallma k oncogene MYC d i es he
p og ession o mos umou s, bu di ec inhibi ion
o MYC by a small- molecule d ug has no eached
clinical es ing. MYC is a ansc ip ion ac o ha
depends on se e al binding pa ne s o unc ion. We
he e o e explo ed he possibili y o a ge ing MYC ia
i s in e ac ome in panc ea ic duc al adenoca cinoma
(PDAC).
Design To iden i y he mos sui able a ge s among all
MYC binding pa ne s, we cons uc ed a a ge ed shRNA
lib a y and pe o med sc eens in cul u ed PDAC cells and
umou s in mice.
Resul s Unexpec edly, many MYC binding pa ne s
we e ound o be impo an o cul u ed PDAC cells
bu dispensable in i o. Howe e , some we e also
essen ial o umou s in hei na u al en i onmen and,
among hese, he ATPases RUVBL1 and RUVBL2 anked
i s . Deg ada ion o RUVBL1 by he auxin- deg on
sys em led o he a es o cul u ed PDAC cells bu no
un ans o med cells and o comple e umou eg ession
in mice, which was p eceded by immune cell in il a ion.
Mechanis ically, RUVBL1 was equi ed o MYC o
es ablish oncogenic and immunoe asi e gene exp ession
iden i ying he RUVBL1/2 complex as a d uggable
ulne abili y in MYC- d i en cance .
Conclusion One implica ion o ou s udy is ha
PDAC cell dependencies a e s ongly in luenced by he
en i onmen , so gene ic sc eens should be pe o med
in i o and in i o. Mo eo e , he auxin- deg on sys em
can be applied in a PDAC model, allowing a ge
alida ion in li ing mice. Finally, by e ealing he nuclea
unc ions o he RUVBL1/2 complex, ou s udy p esen s
a pha maceu ical s a egy o ende panc ea ic cance s
po en ially suscep ible o immuno he apy.
INTRODUCTION
Ad anced solid umou s a e s ill incu able in
many cases, despi e he success ul de elopmen
o a ge ed cance he apies in ecen yea s.1 An
impo an example whe e only limi ed he apeu ic
p og ess has been made is panc ea ic duc al adeno-
ca cinoma (PDAC), a cance ha de i es om he
epi helial exoc ine panc eas. Pa ien s wi h PDAC
ha e a median su i al o less han 1 yea ,2 esul ing
in 530 000 dea hs pe yea wo ldwide.
The main limi a ion o a ge ed cance he apy,
in gene al and pa icula ly in PDAC, is i s na ow
applicabili y. Indeed, each a ge ed he apy is
designed o a speci ic subse o pa ien s whose
umou s ha e speci ic gene ic p o iles.3 While he
WHAT IS ALREADY KNOWN ON THIS TOPIC
⇒Dozens o MYC binding pa ne s ha e been
iden i ied, bu no sys ema ic analysis has
in es iga ed hei sui abili y as cance a ge s
in a eal umou con ex . Ou sc eening hi
RUVBL1 is a case in poin . While i has been
shown ha RUVBL1 is essen ial o MYC-
media ed ancho age- independen g ow h
o ib oblas s, i emained unclea whe he
RUVBL1 is an essen ial e ec o p o ein
o MYC in es ablished panc ea ic duc al
adenoca cinoma (PDAC) in i o and hus a
iable he apeu ic a ge .
WHAT THIS STUDY ADDS
⇒Ou wo k has shown ha only a ew MYC
binding pa ne s a e essen ial o PDAC
p og ession in mice. One o hem, RUVBL1,
is equi ed o he ac i a ion o MYC- d i en
gene exp ession and immune e asion. Acu e
deple ion o RUVBL1 by he auxin- deg on
sys em induced apid e adica ion o PDAC
umou s, accompanied by a signi ican
in il a ion o CD3- posi i e immune cells.
HOW THIS STUDY MIGHT AFFECT RESEARCH,
PRACTICE OR POLICY
⇒Ad anced PDAC emains incu able despi e
ecen ad ances in a ge ed he apies and
immuno he apy. The iden i ica ion o RUVBL1
as a a ge in MYC- d i en PDAC umou s
and ou obse a ion ha RUVBL1 deple ion
sensi ises o immune checkpoin inhibi o s
opens a he apeu ic s a egy, as his AAA
ATPase could be conside ed d uggable by small-
molecule inhibi o s.
copy igh . on Sep embe 9, 2024 a Uni e si a sbiblio hek Zei sch i enab eilung. P o ec ed byh p://gu .bmj.com/Gu : i s published as 10.1136/gu jnl-2023-331519 on 31 May 2024. Downloaded om
1510 Vog M, e al. Gu 2024;73:1509–1528. doi:10.1136/gu jnl-2023-331519
Panc eas
a ailabili y o a a ge ed he apy is an a ac i e he apeu ic
op ion o his pa icula pa ien subg oup, he co esponding
gene ic mu a ion is o en a e, so he numbe o pa ien s who can
bene i is small. This conund um also applies o immuno he a-
pies, whe e only pa ien s wi h an immunologically ac i e umou
mic oen i onmen bene i om immune checkpoin blockade.4
To da e, i has p o en impossible o de elop a uni e sally appli-
cable a ge ed cance he apy by inhibi ing common oncogenes.
An impo an example o oncogenes wi h uni e sal signi -
icance is he amily o MYC ansc ip ion ac o s, which
comp ises c- MYC (called MYC he ea e ), NMYC and LMYC.5 6
Se e al lines o expe imen al and clinical esea ch sugges ha
MYC should be an ou s anding cance a ge . Mos umou s
ha e de egula ed MYC exp ession caused by a ious mecha-
nisms including soma ic gene ampli ica ions and ansloca ions,
mu a ions ha inc ease MYC p o ein s abili y and al e a ions in
ups eam pa hways ha enhance MYC ansc ip ion.7 The link
be ween high MYC exp ession and umo igenesis is causa i e,
since exogenous exp ession o MYC in mouse models ini ia es8
o accele a es9 umou de elopmen . Impo an ly, de egula ed
MYC exp ession d i es umou ini ia ion and ende s es ablished
umou s ‘addic ed’ o i , since silencing exogenous MYC i e e s-
ibly e adica es umou cells.10–14 Mo eo e , sys emic exp ession
o a dominan nega i e allele o MYC, called Omomyc, induced
eg ession o es ablished mu ine umou s bu was ole a ed by
no mal cells,15 sugges ing he exis ence o a he apeu ic window
o he pha maceu ical inac i a ion o MYC. S ikingly, pe i-
odic inhibi ion o endogenous MYC by Omomyc pe manen ly
p e en ed umou p og ession in mice wi hou any sign o esis-
ance.16 Recombinan Omomyc showed he apeu ic e icacy in
p eclinical models o lung cance 17 and has been es ed o sa e y
in a phase I clinical ial (NCT04808362).18
Clinical es ing o an MYC- de i ed he apeu ic p o ein is
exci ing, bu i migh be di icul o deli e mac omolecules o
umou s in o gans such as he b ain and panc eas.19 Indeed,
p o ein- based d ugs ha e mo e limi ed d ug- deli e y p ope -
ies compa ed wi h small- molecule d ugs, bu so a no small-
molecule inhibi o o MYC has ad anced o clinical es ing o
a ious easons. MYC lacks ca aly ic ac i i y ha can be easily
inhibi ed; ins ead, MYC unc ions by binding o he p o eins and
DNA. The DNA- binding domain o MYC is o med by ex ensi e
in e ac ions wi h i s pa ne MAX20 and is s uc u ally simila
o he co esponding domains o o he basic helix- loop- helix
ansc ip ion ac o s. The ansac i a ion domain o MYC is
in insically diso de ed. These s uc u al ea u es impede he
de elopmen o small- molecule d ugs ha di ec ly inhibi MYC.
An a ac i e al e na i e o inhibi ing MYC di ec ly is
a ge ing a binding pa ne ha is a be e d ug a ge han MYC
i sel . MYC’s DNA binding and ansac i a ion ac i i ies bo h
depend on MYC binding pa ne s, as has been explici ly shown
o MAX, WDR5, PAF, SPT5 and TRRAP.21–28 Howe e , MYC
has b oad a ini y o o he p o eins, and dozens o pu a i e
binding pa ne s ha e been iden i ied by sys ema ic p o eomics
me hods.28–31 I is cu en ly unclea which o hem a e essen ial
o MYC unc ion and oncogenic g ow h in PDAC. We he e-
o e used a a ge ed shRNA lib a y o analyse he dependence
o panc ea ic cance cells on 91 MYC binding pa ne s, in i o
and in i o. Ou gene ic sc eens e ealed ha among he mos
essen ial MYC binding pa ne s we e RUVBL1 and RUVBL2,
which a e AAA ATPases ha o m he e ome ic complexes. We
obse ed ha oncogenic MYC exp ession ende s cells depen-
den on he ac i i y o he RUVBL1/2 complex and ha acu e
deple ion o inhibi ion o RUVBL1 p o okes immune in il a-
ion and e adica es panc ea ic umou s in mice.
RESULTS
Gene ic d opou sc eens e eal di e en ial dependence on
MYC binding pa ne s in PDAC in i o and in i o
To assess he essen iali y o MYC binding pa ne s in PDAC, we
gene a ed a doxycycline- inducible shRNA lib a y (con aining
i e shRNAs pe gene and 18 non- a ge ing con ol (NTC)
shRNAs)32 agains 91 MYC binding pa ne s iden i ied in quan-
i a i e mass spec ome y (MS) expe imen s o na i ely isola ed
MYC complexes. Many o he candida es ha e been iden i ied
mul iple imes in addi ional published in e ac ion s udies28–31
(online supplemen al able 1). PCR ampli ica ion o he lib a y
(online supplemen al igu e S1A,B) ollowed by sequencing iden-
i ied all 478 expec ed shRNAs. The abundance o 93.3% o he
shRNAs di e ed by less han 10- old, e i ying hei e en dis i-
bu ion wi hin he lib a y (online supplemen al igu e S1C).33
The shRNA lib a y was used o gene ic d opou sc eening
in bo h in i o and in i o PDAC models ( igu e 1A). Fi s ,
he oncogenic po en ial o he 91 MYC binding pa ne s was
es ed in he mu ine KPC cell line ha bou ing oncogenic mu a-
ions in KRAS and p53,34 because his cell line eng a s in
C57BL/6J mice and o ms MYC- dependen umou s.35 KPC
cells we e ansduced wi h he lib a y, hen spli and cul u ed
wi hou o wi h doxycycline o ac i a e shRNA exp ession. A e
14 days, genomic DNA was isola ed and sequenced o iden i y
in eg a ed shRNAs in he wo ea men g oups. This analysis
showed ha o each shRNA he changes we e simila among
h ee eplica es, and o each MYC binding pa ne he changes
we e simila among he i e shRNAs, documen ing he obus -
ness o he sc eening sys em ( igu e 1B). Fu he mo e, i demon-
s a ed ha exp ession o shRNAs agains 61 o he a ge s led
o an a leas wo old d opou , sugges ing hei essen iali y o
KPC cell g ow h (online supplemen al able 2). We wonde ed
whe he he dependence o KPC cells on speci ic MYC binding
pa ne s di e s in cul u ed cance cells o o he umou ypes.
When we calcula ed an ‘essen iali y sco e’ (pe cen age o 1086
cance cell lines in DepMap o which a gene is essen ial),36 we
obse ed ha 54 o he 61 pa ne s we e essen ial in >50% o all
es ed cance cell lines. Only he candida e ATAD3A, which also
exe s mi ochond ial unc ions, shows s ong de ia ions om
he DepMap da abase, which may be ela ed o syn he ic e ec s
wi h he doxycycline used in ou sc een.37 Taken oge he , hese
esul s sugges ha he iabili y o cul u ed KPC cells depends
on he majo i y o MYC binding pa ne s, o which mos a e also
essen ial o cul u ed cells o o he umou ypes.
Since condi ions in cell cul u e a e undamen ally di e en
om hose in a li ing o ganism, we in es iga ed whe he he
dependence o panc ea ic cance cells on MYC binding pa ne s is
di e en in a umou con ex . We se up an in i o gene ic sc een
using shRNA- ansduced KPC cells o ho opically eng a ed
in o he panc eas o immunocompe en mice ( igu e 1A). A
i s expe imen e ealed ha an injec ion o 100 000 cells was
needed o achie e uni o m ep esen a ion o he lib a y (online
supplemen al igu e S1C). We he e o e eng a ed 10 mice wi h
100 000 cells each, induced shRNA exp ession by doxycycline in
i e o he animals and analysed in eg a ed shRNA abundance in
umou s a e 14 days.
Fi s , we compa ed he abundance o in eg a ed shRNA in
he i e umou s o he un ea ed mice wi h ha in he o igi-
nally ansduced KPC cells ( igu e 1C). No shRNA was ound
o be signi ican ly deple ed in umou s, con i ming ha shRNA
exp ession is no induced in he absence o doxycycline. In a
compa ison be ween doxycycline- ea ed and un ea ed mice, 70
o he 460 a ge ing shRNAs we e signi ican ly deple ed (log2
copy igh . on Sep embe 9, 2024 a Uni e si a sbiblio hek Zei sch i enab eilung. P o ec ed byh p://gu .bmj.com/Gu : i s published as 10.1136/gu jnl-2023-331519 on 31 May 2024. Downloaded om
1511
Vog M, e al. Gu 2024;73:1509–1528. doi:10.1136/gu jnl-2023-331519
Panc eas
Figu e 1 Gene ic d opou sc eens e eal di e en ial dependence on MYC binding pa ne s in panc ea ic duc al adenoca cinoma (PDAC) in i o and
in i o. (A) Schema ic o he in i o (KPC cul u e) and in i o (KPC allog a ) d opou sc eens o 91 MYC binding pa ne s. (B) Wa e all plo o he
in i o sc een in KPC cells showing he doxycycline (Dox)- induced changes in abundance o 478 shRNAs (log2FC±SEM, n=3). shRNAs a e g ouped
by a ge gene, so ed by he median change o he i e shRNAs. Selec ed genes o in e es , including MYC, a e ma ked. The essen iali y sco e is he
pe cen age o human cance cell lines in he DepMap po al ha depend on each gene o iabili y. NTC1–4 co espond o g oups o ou o i e non-
a ge ing con ol shRNAs. (C) Volcano plo o he in i o sc een showing changes in abundance (en ichmen ) o 460 a ge ing shRNAs (black do s)
and 18 non- a ge ing shRNAs (g een do s), be ween i e umou s om mice ed s anda d chow (he e, ‘Vehicle’) and ansplan ed KPC cells (Inpu ).
Wald es p alues o i e mice. (D) Volcano plo o he in i o sc een showing changes in abundance (en ichmen ) o 460 a ge ing shRNAs (black
do s) and 18 non- a ge ing shRNAs (g een do s), be ween i e umou s om mice ed doxycycline- con aining chow (Dox) and i e umou s om mice
ed s anda d chow (Vehicle). Wald es p alues. (E) Wa e all plo o he in i o sc een compa ing in eg a ed shRNA abundance in umou s o i e
mice ed doxycycline- con aining chow (Dox) and i e mice ed s anda d chow (Vehicle). Ba s ep esen he median log2FC o i e shRNAs pe gene,
and e o ba s ep esen he SEM. (F) Hea map o he in i o sc een. The ch oma ic scale indica es he median change in in eg a ed shRNA abundance
(log2FC) pe gene ela i e o ehicle- ea ed mice. Each column co esponds o one mouse ed ei he s anda d chow (Vehicle) o doxycycline-
con aining chow (Dox). Genes a e so ed by he median change o i e Dox- ea ed mice. (G) Sca e plo compa ing he esul s o he in i o
(cul u ed KPC cells) and in i o (allog a ed KPC cells) sc eens. Do s ep esen he median change (log2FC) o i e shRNAs pe gene and a e colou
coded acco ding o he essen iali y sco e. NTC1–4, g oups o ou o i e NTC shRNAs. (H) Sca e plo as in panel G. Red, TIP60 complex componen s.
G een, NTC shRNAs. (I) Sca e plo as in panel G. Red, genes ha a e mo e essen ial in KPC cells han in NIH3T3 cells ( alues a e epo ed in online
supplemen al able 2). G een, NTC shRNAs. See also online supplemen al igu e S1.
copy igh . on Sep embe 9, 2024 a Uni e si a sbiblio hek Zei sch i enab eilung. P o ec ed byh p://gu .bmj.com/Gu : i s published as 10.1136/gu jnl-2023-331519 on 31 May 2024. Downloaded om
1512 Vog M, e al. Gu 2024;73:1509–1528. doi:10.1136/gu jnl-2023-331519
Panc eas
old change (log2FC) <−1, p<0.05), while he abundance o
he 18 NTC shRNAs was unchanged ( igu e 1D). When we
examined he median shRNA change pe gene, we obse ed
ha only 26 MYC binding pa ne s we e signi ican ly deple ed
( igu e 1E,F).
To unde s and how he dependency o KPC cells di e s
be ween he in i o and in i o condi ions, we compa ed he
doxycycline- induced change in all MYC binding pa ne s in a
sca e plo ( igu e 1G; see also online supplemen al able 2).
This analysis e ealed ha 31 MYC binding pa ne s, such as
he SMARCD2 subuni o he SWI/SNF ch oma in emodelling
complex, a e no essen ial in ei he condi ion. Fu he mo e, 34
MYC binding pa ne s we e ound o be essen ial in i o bu
dispensable in i o. This g oup con ained many p o eins ha
a e conside ed common essen ial genes in DepMap, including
p o eins ha a e conside ed oncogenic a ge s such as he an-
sc ip ion ac o s YY138 and p400,39 and he cohesin complex
membe RAD21.40 Rema kably, he cons i u i e binding pa ne
o MYC’s DNA binding domain, MAX,5 41 was also no essen-
ial in i o. We hypo hesise ha pa ial deple ion o MAX is
neu al in panc ea ic umou s, since MAX also o ms ep es-
si e homodime s and he e odime s ha bind o simila DNA
mo i s and compe e wi h he gene- ac i a ing capaci y o he
MYC/MAX he e odime s. This obse a ion is also consis en
wi h he dominan - nega i e unc ion o Omomyc, which shi s
MAX om ac i a ing MYC/MAX o ansc ip ionally inac i e
Omomyc/Max dime s.15 16 42 43
Finally, 27 genes we e ound o be essen ial bo h in i o
and in i o. Among hese p o eins a e MYC i sel and i s well-
cha ac e ised pa ne s WDR5,23 TOP2A44 and SPT6.28 S ikingly,
his g oup also included ou membe s o he TIP60 complex,
namely RUVBL1, RUVBL2, TRRAP and ACTL6A ( igu e 1H).
To de e mine i he e a e MYC binding pa ne s ha a e essen-
ial o PDAC umou s bu dispensable o non- cance cells,
we epea ed he d opou sc een in he NIH3T3 ib oblas cell
line. This analysis showed ha 51 MYC binding pa ne s a e
essen ial o he g ow h o hese un ans o med cells (log2FC
<−1) (online supplemen al able 2). A compa ison wi h KPC
cells e ealed ha only RUVBL1, RUVBL2, TRRAP, SPT6 and
WDR5 we e essen ial in panc ea ic umou s and mo e impo an
o KPC cells han ib oblas s (Δlog2FC<0, igu e 1I). We
concluded ha (1) he dependencies o panc ea ic cance cells on
MYC binding pa ne s di e ed in i o and in i o and ha (2)
i e candida es we e less essen ial o ib oblas han o PDAC
umou s.
Exp ession o MYC and RUVBL1 co ela e in PDAC and high
le els a e associa ed wi h agg essi e umou s
Nex , we wan ed o ind ou which MYC e ec o p o eins a e
highly exp essed in PDAC umou s wi h high MYC ac i i y, bu
show low exp ession in umou s wi h low MYC le els and in
no mal issue. Fo his pu pose, we i s analysed he co- exp es-
sion o all 91 candida es wi h MYC in umou s om pa ien s wi h
PDAC collec ed in he cu a ed TCGA da abase (n=159).45 We
co ela ed he RNA exp ession o MYC a ge genes (as a p oxy
o MYC ac i i y) wi h he RNA exp ession o e e y candida e
in PDAC p ima y umou samples (online supplemen al igu e
S2A, online supplemen al able 3). The exp ession o 25 MYC
binding pa ne s co ela ed s ongly wi h MYC le els (Pea son’s
co ela ion coe icien >0.5). We hen compa ed he exp ession
o all MYC binding pa ne s in PDAC umou s o hei exp es-
sion in heal hy panc ea ic issue (n=4, online supplemen al
igu e S2B, online supplemen al able 3) e ealing ha 25 MYC
binding pa ne s a e o e exp essed in PDAC samples compa ed
wi h heal hy issue (log2FC>0.3).
The only MYC binding pa ne s ha (1) showed deple ion
in he in i o sc een, (2) had a good co ela ion be ween hei
exp ession and MYC ac i i y in PDAC umou s ( igu e 2A) and
(3) we e o e exp essed in PDAC umou s compa ed wi h heal hy
panc ea ic issue we e he p o eins RUVBL1 and RUVBL2, wi h
RUVBL1 sco ing he highes . We alida ed inc eased RUVBL1
exp ession in pa ien s wi h human cance a he p o ein le el
using an in- house issue mic oa ay (TMA) con aining PDAC
umou , benign acina and benign duc al issue specimens s ained
o RUVBL1 ( igu e 2B,C, online supplemen al igu e S2C,D).
S ikingly, pa ien s wi h high le els o MYC and RUVBL1 show
signi ican ly educed o e all su i al compa ed wi h pa ien s
wi h low exp ession ( igu e 2D, online supplemen al igu e S2E).
In e es ingly, RUVBL1 exp ession is highe in basal and undi e -
en ia ed umou s han in glandula and di e en ia ed umou s,
espec i ely (online supplemen al igu e S2F,G). We concluded
ha pa ien s wi h PDAC wi h ele a ed MYC exp ession also
o e exp ess RUVBL1, and ha umou s wi h he highes exp es-
sion o bo h p o eins a e he mos agg essi e.
RUVBL1 is essen ial o DNA eplica ion and g ow h o
panc ea ic cance cells
RUVBL1 and RUVBL2 a e AAA ATPases ha o m he e ome ic
hexame s o dodecame s (online supplemen al igu e S3A).46 In
cells, hey a e subuni s o se e al mul ip o ein complexes, such
as he R2TP/PAQosome chape one complex, he his one ace -
yl ans e ase complex TIP60 and he ch oma in emodelling
complex INO80. Mo eo e , ecen ly hei ole in RNA poly-
me ase II (RNAPII) clus e ing has been desc ibed.47 We aimed
o es ablish a a ge ed p o ein deple ion sys em o he apid
deg ada ion o RUVBL1 o s udy he di ec oncogenic unc-
ion o he RUVBL1/2 complex in PDAC. Fo his pu pose, we
inse ed he auxin- inducible deg on (AID) sequence in o he
Ru bl1 locus in KPC cells ( igu e 3A, online supplemen al igu e
S3B,C). Immunoblo ing con i med he success ul usion o he
AID ag wi h RUVBL1 and he lack o exp ession o wild- ype
RUVBL1 ( igu e 3B). We hen s ably exp essed in hese cells he
plan - de i ed E3 ligase TIR1F74G ha , in he p esence o auxin
(5- phenyl- 1H- indole- 3- ace ic acid), enables he deg ada ion o
AID- agged p o eins a low auxin concen a ions.48 Acco dingly,
we obse ed deg ada ion o RUVBL1 in KPCAID- Ru bl1; TIR1 cells
a nanomola concen a ions and comple e deple ion a 1 µM
auxin ( igu e 3C). S ikingly, incuba ion o KPCAID- Ru bl1; TIR1 cells
wi h auxin o 6 hou s dec eased RUVBL1 o unde ec able le els
( igu e 3D,E).
Auxin- media ed deple ion o RUVBL1 in KPCAID- Ru bl1; TIR1
cells subs an ially s opped cell g ow h, while auxin had no
impac on he g ow h o KPCAID- Ru bl1 cells wi hou TIR1F74G
( igu e 3F). To unde s and why he cells a es ed on RUVBL1
deple ion, we analysed he cell cycle dis ibu ion by B dU- PI
low cy ome y ( igu e 3G–I, online supplemen al igu e S3D).
Auxin- media ed deple ion o RUVBL1 educed he numbe o
B dU- posi i e cells a 24 hou s and comple ely p e en ed any
B dU inco po a ion a e 48 hou s. Simila e ec s we e obse ed
wi h CB- 6644, an allos e ic RUVBL1/2 inhibi o 49 ( igu e 3G–I,
online supplemen al igu e S3D). These esul s sugges ha
RUVBL1/2 complex o ma ion o ca aly ic ac i i y is c ucial
o he g ow h- p omo ing unc ion o RUVBL1. Mo eo e , he
obse ed a es in S- phase on deple ion o inhibi ion o RUVBL1
was compa able o wha was desc ibed on silencing MYC in
panc ea ic cance cells.35
copy igh . on Sep embe 9, 2024 a Uni e si a sbiblio hek Zei sch i enab eilung. P o ec ed byh p://gu .bmj.com/Gu : i s published as 10.1136/gu jnl-2023-331519 on 31 May 2024. Downloaded om
1513
Vog M, e al. Gu 2024;73:1509–1528. doi:10.1136/gu jnl-2023-331519
Panc eas
RUVBL1 edi ec s ansc ip ion om immune genes o g ow h
genes
To de e mine i RUVBL1 di ec ly in luences ansc ip ion, we
combined auxin- media ed deple ion wi h SLAM- seq, a me hod
o iden i y new ansc ip s by 4sU labelling. We ea ed KPCAID-
Ru bl1; TIR1 cells wi h auxin o 3 o 15 hou s o deple e RUVBL1
and labelled newly syn hesised ansc ip s wi h 4sU o 2 hou s
(online supplemen al igu e S4A). The ansc ip ional conse-
quences o 3 hou s o RUVBL1 deple ion we e simila o hose
o 15 hou s, bu he o e all impac was sligh ly weake wi h
some ansc ip s being a ec ed di e en ly (online supplemen al
igu e S4B, online supplemen al able 4).
We used gene se en ichmen analysis o analyse RUVBL1-
media ed ansc ip ional consequences and he unde lying
kine ics. S ikingly, ansc ip s o known MYC a ge genes we e
down egula ed (ie, ac i a ed by RUVBL1) a bo h ime poin s
and we e o e all he mos s ongly a ec ed genes ( igu e 4A–C,
online supplemen al able 5). This g oup o genes includes a se
o MYC- induced ansc ip s iden i ied ea lie , in an AID deple-
ion sys em o MYC, as p ima y MYC a ge genes.50 The mos
up egula ed genes (hence ep essed by RUVBL1) a e acu e
RUVBL1 deple ion encode p o eins media ing immune signal-
ling, such as componen s o he umou nec osis ac o alpha,
ans o ming g ow h ac o - be a and in e e on-γ-signalling
pa hways ( igu e 4A–C); hese genes ha e al eady been shown
o be ep essed by MYC in a ious sys ems.12–14 35 51 T ea men
o KPC cells wi h he RUVBL1/2 complex inhibi o CB- 6644
o 24 hou s induced simila ansc ip ional changes as did
RUVBL1 deple ion ( igu e 4D, online supplemen al igu e S4C).
To es whe he ac i a ion o MYC a ge genes and ep ession
o immune genes is a gene al unc ion o RUVBL1 in panc e-
a ic cance cells, we ea ed i e u he mu ine PDAC lines
wi h di e en gene ic backg ounds wi h CB- 6644 o 24 hou s
and analysed gene exp ession by RNA sequencing. S ikingly,
we obse ed a obus up egula ion o in e e on signalling and
down egula ion o MYC a ge genes in all cell lines es ed
(online supplemen al igu e S4D,E).
We wonde ed i RUVBL1 egula es MYC a ge genes by
binding o hei p omo e s. The e o e, we s udied RUVBL1 and
MYC binding o ch oma in globally in KPCAID- Ru bl1; TIR1 cells
Figu e 2 Exp ession o MYC and RUVBL1 co ela e in panc ea ic duc al adenoca cinoma (PDAC) and high le els a e associa ed wi h agg essi e
umou s. (A) Sca e plo compa ing exp ession o RUVBL1 and MYC a ge gene exp ession (mean o all HALLMARK MYC TARGET V1 genes a e
scaling exp ession (FPKM) ac oss all TCGA pa ien s) in pa ien s wi h human PDAC om he TCGA da abase ( , Pea son’s co ela ion coe icien ; p
alue, unpai ed - es , n=159). (B) Exempla y immunohis ochemis y o RUVBL1 om a issue co e om a issue mic oa ay con aining 31 indi idual
human PDAC specimens. A sec ion wi h high RUVBL1 exp ession and a zoom- in wi h PDAC (P) and s omal issue (S) a e shown (scale: 200 µm). The
panel is also shown as pa o online supplemen al igu e S2C. (C) Quan i ica ion o RUVBL1 exp ession in a issue mic oa ay con aining 31 sec ions
o human PDAC specimens and 24 sec ions o benign acina issue as in panel B. RUVBL1 exp ession was sco ed as nega i e, low, medium and high
in PDAC, adjacen s oma and non- malignan duc al and acina issue. The a io o issues wi h high RUVBL1 exp ession is shown. n, sample size. (D)
Kaplan- Meie su i al cu es o pa ien s wi h PDAC s a i ied in o g oups o low and high exp ession o RUVBL1 and MYC a ge genes (mean o all
MYC TARGET V1 genes a e scaling exp ession (FPKM) ac oss all TCGA pa ien s). P alue, log- ank es . See also online supplemen al igu e S2.
copy igh . on Sep embe 9, 2024 a Uni e si a sbiblio hek Zei sch i enab eilung. P o ec ed byh p://gu .bmj.com/Gu : i s published as 10.1136/gu jnl-2023-331519 on 31 May 2024. Downloaded om

1514 Vog M, e al. Gu 2024;73:1509–1528. doi:10.1136/gu jnl-2023-331519
Panc eas
ea ed wi h ehicle o auxin by ch oma in immune p ecipi-
a ion (ChIP) ollowed by sequencing. In ehicle- ea ed cells,
RUVBL1 occupied 10 016 genomic si es, o which 7386 we e
in RNAPII p omo e s (online supplemen al igu e S4F). Auxin-
media ed deple ion o RUVBL1 d as ically educed he numbe
o RUVBL1- occupied si es o 805 and b ough he signals a
p omo e s o backg ound le els ( igu e 4E,F). MYC occu-
pied 15 991 genomic si es, o which 7906 we e in p omo e s
(online supplemen al igu e S4F). Since he absolu e numbe o
peaks s ongly depends on he chosen peak calling algo i hm
and h esholds (online supplemen al igu e S4G), and since
bo h MYC and RUVBL1 show a s ong p omo e p e e ence
( igu e 4G), we quan i ied he occupancy o bo h p o eins a
all anno a ed p omo e s and obse ed a s iking co ela ion
( =0.913, igu e 4H, online supplemen al igu e S4H).
We wonde ed i p omo e occupancy o MYC and RUVBL1
co ela es wi h he ansc ip ion changes due o acu e RUVBL1
deple ion. We so ed all ansc ip s iden i ied in he SLAM- seq
Figu e 3 RUVBL1 is essen ial o DNA eplica ion and g ow h o panc ea ic cance cells. (A) Ru bl1 knock- in s a egy o auxin- inducible deg on
(AID) agging showing he elemen s o he knock- in casse e and he a chi ec u e o Ru bl1. A ows indica e he posi ion o p ime s used o iden i y
ecombined cell clones by PCR (see online supplemen al igu e S3B). (B) Immunoblo s o wild- ype KPC and KPCAID- Ru bl1 cell lysa es p obed wi h
an ibodies agains RUVBL1 o he AID ag. (C) Immunoblo o KPCAID- Ru bl1; TIR1 cells ea ed wi h a ious concen a ions o auxin o 3 o 6 hou s.
Vinculin, loading con ol. (D) Immunoblo o KPCAID- Ru bl1; TIR1 cells ea ed wi h 1 µM auxin o e ime. TIR1F74G was de ec ed wi h an an ibody agains
he MYC ag. Vinculin, loading con ol. (E) Quan i ica ion o immunoblo s o RUVBL1 a e 6 hou s o 1 µM auxin ea men (n=5, mean±SD, unpai ed
- es ). U, ela i e uni s. (F) Loga i hmic g ow h cu e o KPCAID- Ru bl1 cells exp essing o no exp essing TIR1F74G. Cells we e ea ed daily wi h 1 µM
auxin o ehicle, and g ow h was ollowed o 9 days in biological iplica es (n=3, mean±SD). (G) Immunoblo o KPCAID- Ru bl1; TIR1 cells ea ed wi h 1
µM auxin o 1 µM CB- 6644 o indica ed ime poin s. Vinculin, loading con ol. (H) B dU- PI low cy ome y sca e plo s o KPCAID- Ru bl1; TIR1 cells a e
ea men wi h 1 µM auxin o 1 µM CB- 6644 o 24 o 48 hou s. Cells we e labelled wi h B dU o 1 hou . (I) Quan i ica ion o S- phase cells wi h high
o low B dU inco po a ion as shown in panel H. The expe imen was pe o med in biological duplica es (n=2, mean). See also online supplemen al
igu e S3.
copy igh . on Sep embe 9, 2024 a Uni e si a sbiblio hek Zei sch i enab eilung. P o ec ed byh p://gu .bmj.com/Gu : i s published as 10.1136/gu jnl-2023-331519 on 31 May 2024. Downloaded om
1515
Vog M, e al. Gu 2024;73:1509–1528. doi:10.1136/gu jnl-2023-331519
Panc eas
Figu e 4 RUVBL1 edi ec s ansc ip ion om immune genes o g ow h genes. (A) Gene se en ichmen analysis (GSEA) plo s o selec ed RUVBL1-
ac i a ed and RUVBL1- ep essed gene se s. GSEA was pe o med on SLAM- seq da a o KPCAID- RUVBL1; TIR1 cells. Cells we e ea ed wi h 1 µM auxin o
DMSO (Vehicle) o 15 hou s, ollowed by 800 µM 4sU o 2 hou s. The no malised en ichmen sco e (NES) is posi i e o gene se s ac i a ed by and
nega i e o hose ep essed by RUVBL1 (FDR, alse disco e y a e). (B) GSEA o KPCAID- Ru bl1; TIR1 cells ea ed wi h 1 µM auxin o DMSO o 15 hou s
and 800 µM 4sU. The NES and he q- alue a e shown o all en iched gene se s (posi i e NES: gene se s ac i a ed by RUVBL1, nega i e NES: gene
se s ep essed by RUVBL1). (C) NES alues o di e en hallma k gene se s and a gene se con aining p ima y MYC a ge s de ined in Muha e al,50
compa ed be ween he 3 and 15- hou du a ions o RUVBL1 deple ion. (D) Sca e plo compa ing gene egula ion a e auxin- induced deg ada ion
and CB- 6644 inhibi ion o RUVBL1. KPCAID- Ru bl1; TIR1 cells we e ea ed wi h 1 µM auxin o 15 hou s (n=3) o 1 µM CB- 6644 o 24 hou s (n=3).
Gene exp ession was analysed by SLAM- seq. Changes (log2FC) in o al RNA e sus DMSO ( ehicle)- ea ed cells a e shown ( , Pea son’s co ela ion
coe icien ; p alue, unpai ed - es ). (E) Genome b owse ack o RUVBL1 ch oma in immune p ecipi a ion (ChIP)- seq signal in KPCAID- Ru bl1; TIR1 cells
ea ed wi h ehicle o 1 µM auxin o 15 hou s. Binding o he Rpl8 gene is shown as spike- no malised eads and compa ed wi h he inpu signal as
con ol. (F) Densi y plo s o RUVBL1 ChIP- seq signals a ound ansc ip ion s a si es (TSS). A e aged binding (RPKM) o RUVBL1 in KPCAID- Ru bl1; TIR1
cells ea ed wi h DMSO ( ehicle) o 1 µM auxin o 15 hou s compa ed wi h he inpu signal. (G) Ba g aph o he genomic dis ibu ion o RUVBL1
and MYC peaks. Ch oma in binding o RUVBL1 and MYC was analysed by ChIP- seq and was compa ed wi h a se o andom genomic in e als in
p omo e s (TSS− 3000 bp o TSS+ 3000 bp), gene bodies (TSS+ 3000 bp o ansc ip ion end si e (TES)), egions downs eam o genes (TES o TES+
2000 bp) and in e genic egions. (H) Sca e plo compa ing he p omo e occupancy o MYC and RUVBL1 (spike- no malised eads) as measu ed
by ChIP- seq. , Pea son’s co ela ion coe icien ; p alue, unpai ed - es . (I) Bin plo compa ing gene egula ion due o acu e RUVBL1 deple ion
wi h RUVBL1 and MYC binding (spike- no malised eads) a gene p omo e s. Genes we e binned in o eigh equally dis an bins o gene egula ion
on 15 hou s o RUVBL1 deple ion (‘Regula ion’). Mean egula ion pe bin was plo ed agains mean p omo e occupancy by MYC and RUVBL1 in
unpe u bed KPCAID- Ru bl1; TIR1 cells (mean±SEM). , Pea son’s co ela ion coe icien ; p alue, unpai ed - es . NFkB, nuclea ac o kappa B; TGFβ,
ans o ming g ow h ac o - be a; TNFα, umou nec osis ac o alpha. See also online supplemen al igu e S4.
copy igh . on Sep embe 9, 2024 a Uni e si a sbiblio hek Zei sch i enab eilung. P o ec ed byh p://gu .bmj.com/Gu : i s published as 10.1136/gu jnl-2023-331519 on 31 May 2024. Downloaded om
1516 Vog M, e al. Gu 2024;73:1509–1528. doi:10.1136/gu jnl-2023-331519
Panc eas
expe imen acco ding o hei egula ion on RUVBL1 deple ion
and compa ed his anking o he ex en o p omo e occupancy
by MYC and RUVBL1 in a bin plo ( igu e 4I). These analyses
e ealed ha genes ac i a ed by RUVBL1 ha e highe p omo e
occupancy o bo h MYC and RUVBL1 han non- egula ed and
ep essed genes. We concluded ha RUVBL1 and MYC co- oc-
cupy housands o p omo e s in panc ea ic cance cells and ha
deple ion o inhibi ion o RUVBL1 leads o hei down egula ion.
RUVBL1/2 complex is an essen ial co ac o o MYC
Nex , we wan ed o ind ou whe he RUVBL1 is a c i ical
co ac o o MYC o whe he bo h p o eins bind and egula e
simila genes independen ly. To his end, we i s analysed
whe he RUVBL1 ch oma in binding depends on MYC. We
he e o e aimed o apidly deple e MYC by he auxin- deg on
echnology bu we e unable o in oduce he AID sequence
in o he MYC locus in KPC cells. Ins ead, we could gene a e
he desi ed ansgenic clone in he human melanoma cell line
A375. We he e o e pe o med ChIP- seq expe imen s o
RUVBL1 and MYC in he A375MYC- AID cells (online supplemen al
igu e S5A). Auxin- induced deple ion educed MYC ch oma in
binding o backg ound le els and s ikingly educed RUVBL1
binding a MYC- bound egions ( igu e 5A, online supplemen al
igu e S5B). O e all, RUVBL1 binding was educed in 82.7%
o all join MYC/RUVBL1 peaks, and he deg ee o educ ion
co ela ed wi h he s eng h o MYC binding ( igu e 5B). We
alida ed he loss o RUVBL1 om p omo e s on deple ion o
MYC by ChIP- qPCR expe imen s (online supplemen al igu e
S5C) and concluded ha ch oma in associa ion o RUVBL1
depends on MYC.
To in es iga e i RUVBL1 is c i ical o MYC- media ed gene
egula ion, we combined exogenous o e exp ession o MYC wi h
inhibi ion o he RUVBL1/2 complex. KPC cells we e ansduced
o s ably exp ess MYC- ER, a usion p o ein o MYC and he
oes ogen ecep o ha can be ac i a ed by 4- hyd oxy amoxi en
(OHT). We ea ed cells wi h 200 nM OHT and analysed acu e
Figu e 5 RUVBL1 is an essen ial co ac o o MYC. (A) Genome b owse acks o RUVBL1 and MYC ch oma in immune p ecipi a ion (ChIP)- seq
signal in A375MYC- AID cells ea ed wi h 1 µM auxin o 3 hou s o ehicle con ol. RNA polyme ase II (RNAPII) acks o U2OS cells (GSE162264) a e
shown. Binding o he EIF4A1 and TBRG4 genes is shown as spike- no malised eads and compa ed wi h he inpu signal as con ol. In he TBRG4
p omo e , one o wo RUVBL1 peaks loca ed a an MYC- nega i e egion does no dec ease on auxin- media ed deple ion o MYC. (B) Rank plo o
RUVBL1 and MYC ChIP- seq signal in A375MYC- AID cells ea ed wi h 1 µM auxin o 3 hou s o ehicle con ol. All MYC and RUVBL1- bound p omo e s
a e so ed o dec ease in RUVBL1 ch oma in binding on acu e MYC deple ion and plo ed as log2FC (y- axis, black). Mean MYC binding is plo ed
o 15 equally sized bins con aining he same genes (y- axis, blue). (C) Sca e plo o SLAM- seq da a compa ing MYC- induced gene exp ession
changes in he absence o p esence o CB- 6644. KPCMYC- ER cells we e ea ed wi h DMSO o 1 µM CB- 6644 o 20 hou s ollowed by e hanol o 200
nM 4- hyd oxy amoxi en (OHT) o 4 hou s. Changes in o al RNA (log2FC) a e shown (n=3). The slope (m) and p alue (p) o he linea eg ession
(blue) a e indica ed as is he Pea son’s co ela ion coe icien ( ). A line wi h slope m=1 is shown in black. (D) Hea map o SLAM- seq da a om cells
ea ed as in panel C. Biological eplica es a e labelled 1, 2 and 3. Changes in o al RNA (log2FC) a e shown. (E) Suc ose g adien ul acen i uga ion
demons a ing a physical in e ac ion be ween MYC and RUVBL1/2. Pu i ied ecombinan His6- MBP- MYC1- 163 (MYC1- 163, op panel), RUVBL1/2 (middle
panel) and all h ee p o eins oge he (bo om panel) we e subjec ed o suc ose g adien ul acen i uga ion. F ac ions we e collec ed and analysed by
SDS- PAGE, ollowed by Coomassie blue s aining. AID, auxin- inducible deg on. See also online supplemen al igu e S5.
copy igh . on Sep embe 9, 2024 a Uni e si a sbiblio hek Zei sch i enab eilung. P o ec ed byh p://gu .bmj.com/Gu : i s published as 10.1136/gu jnl-2023-331519 on 31 May 2024. Downloaded om
1517
Vog M, e al. Gu 2024;73:1509–1528. doi:10.1136/gu jnl-2023-331519
Panc eas
changes in ansc ip le els. OHT- media ed ac i a ion o MYC
al e ed gene exp ession in a manne ypical o oncogenic MYC
(online supplemen al igu e S5D,E). S ikingly, incuba ion o
cells wi h he RUVBL1/2 inhibi o CB- 6644 p io o OHT addi-
ion globally a enua ed gene ac i a ion and ep ession by MYC
( igu e 5C,D).
RUVBL1 and RUVBL2 physically and gene ically in e ac
wi h MYC in cells, as demons a ed p e iously by us28 31 and
o he s.52 53 Howe e , MYC also binds di ec ly o he pseudoki-
nase TRRAP,22 which is a pa o he TIP60 complex oge he
wi h RUVBL1 and RUVBL2. To de e mine i MYC binds o he
RUVBL1/2 complex independen ly o TRRAP, we exp essed
MYC1- 163 and RUVBL1 and RUVBL2 in Esche ichia coli (online
supplemen al igu e S5F) and analysed he pu i ied p o eins’
abili y o o m complexes by suc ose g adien ul acen i uga-
ion. When es ed sepa a ely, MYC1- 163 peaked in ac ion 2
and he RUVBL1/2 complex peaked in ac ion 8 ( igu e 5E).
When he p o eins we e incuba ed oge he be o e he assay,
hey peaked in ac ions 8 and 11, espec i ely. This s ong shi
owa ds la e ac ions indica es a di ec in e ac ion be ween he
ecombinan p o eins. A pull- down on ecombinan MYC1- 163
and cop ecipi a ion o RUVBL1/2 con i med a obus in e ac-
ion (online supplemen al igu e S5G).
Nex , we aimed o map he domains ha media e he in e -
ac ion wi h MYC on RUVBL1 o gene a e in e ac ion- de icien
mu an s o RUVBL1. Based on a ecen epo in Ewing
sa coma,54 we designed a se ies o pu a i e loss- o - in e ac ion
mu an s o RUVBL1 (RUVBL1Δ94- 118, RUVBL1Δ102- 107,
RUVBL1K108A) and es ed hei in e ac ion in co- immunop ecip-
i a ion expe imen s in KPC cells. Bo h RUVBL1 mu an s lacking
he loop in he cen al channel o he RUVBL1/2 hexame
(RUVBL1Δ94- 118, RUVBL1Δ102- 107) los he abili y o bind MYC
(online supplemen al igu e S5H) and could no escue he
g ow h de ec caused by auxin- media ed loss o endogenous
RUVBL1 (online supplemen al igu e S5I). We concluded ha
MYC and he RUVBL1/2 complex bind o each o he , explaining
hei co- occupancy on housands o p omo e s and he ele ance
o RUVBL1 o MYC- media ed gene egula ion and g ow h o
panc ea ic cance cells.
RUVBL1 is equi ed o he main enance and p og ession o
panc ea ic cance
Nex , we used he AID sys em o acu ely deple e RUVBL1 in i o
o analyse he e ec s o RUVBL1 on panc ea ic umou g ow h.
To es whe he auxin can each panc ea ic umou s and induce
he deple ion o endogenous a ge p o eins in i o, we ans-
plan ed KPCAID- Ru bl1; TIR1 cells in o C57BL/6J mice panc ea a,
le umou s g ow, adminis e ed a ious doses o auxin and
isola ed umou s a e 6 hou s. Immunoblo ing showed ha
auxin induced he deple ion o AID- agged RUVBL1 bu no o
he wild- ype p o ein exp essed by s omal cells (online supple-
men al igu e S6A). An auxin dose o 1 mg/kg body weigh was
e ec i e, and he highes es ed dose (20 mg/kg) did no cause
isible signs o oxici y. We hen examined RUVBL1 le els in
umou s o e ime a e a single auxin injec ion. A subs an ial
dec ease in AID- agged RUVBL1 was seen a he ea lies ime
poin (2 hou s), and he p o ein s ayed unde ec able o 24 hou s
( igu e 6A). We assumed ha daily adminis a ion o auxin
would esul in du able RUVBL1 deple ion, enabling us o in es-
iga e he ole o RUVBL1 in panc ea ic umou p og ession.
We nex ansplan ed KPCAID- Ru bl1; TIR1 cells in o 14 mice.
Tumou size on day 7, es ima ed by bioluminescence imaging,
was used o g oup mice in o pai s wi h simila size umou s;
one animal o each pai was ea ed daily wi h auxin and
he o he wi h ehicle o 28 days ( igu e 6B). Auxin ea -
men esul ed in dec eased bioluminescence al eady on day 5
and p og essi ely lowe bioluminescence un il day 14, indi-
ca ing ha umou s we e eg essing d as ically ( igu e 6C,D).
Howe e , a la e ime poin s he bioluminescence inc eased,
and umou s e u ned o hei s a ing sizes despi e auxin
ea men . Ins ead, umou s in all ehicle- ea ed animals g ew
p og essi ely om he s a o he expe imen . Su i al anal-
ysis showed ha auxin- induced RUVBL1 deple ion p o ided a
s ong su i al ad an age, wi h a median su i al ime o 47 s
25 days in he ehicle- ea ed g oup ( igu e 6E). To unde s and
why umou s in auxin- ea ed animals es a ed o g ow a e
2 weeks, we isola ed umou s when mice eached he endpoin
and analysed TIR1F74G le els by immunoblo ing ( igu e 6F).
S ikingly, he exp ession o TIR1F74G was d as ically educed
in all auxin- ea ed umou s, indica ing a s ong selec ion o
cells wi hou a unc ional AID sys em in i o. We concluded
ha RUVBL1 is equi ed o he main enance and p og ession
o panc ea ic cance in mice.
Since panc ea ic umou s a e usually de ec ed in pa ien s a
ad anced s ages, we epea ed he ansplan a ion o KPCAID-
Ru bl1; TIR1 cells bu le umou s eng a o 16 ins ead o 7 days
be o e s a ing auxin ea men . While all ehicle- ea ed mice
eached he endpoin wi hin 14 days o ea men , all auxin-
ea ed animals su i ed he ea men pe iod and had a s ong
o e all su i al bene i (online supplemen al igu e S6B,C).
Again, in umou s excised when mice eached he endpoin ,
TIR1F74G exp ession was d as ically educed in all auxin- ea ed
animals (online supplemen al igu e S6D), sugges ing ha e en
ad anced umou s s ic ly depend on RUVBL1.
RUVBL1 p omo es immune e asion in PDAC
We ound i in iguing ha RUVBL1 deple ion igge ed s ong
umou eg ession in i o, whe eas he same umou cells only
a es ed in cul u e. We he e o e examined umou s his ologically
a e 5 days o auxin ea men o s udy he unde lying cellula
mechanisms. Immunohis ochemis y con i med he auxin-
media ed deple ion o RUVBL1 in umou cells ( igu e 7A), while
RUVBL1 le els in he su ounding heal hy issue and s omal
cells we e no a ec ed (online supplemen al igu e S7A). In addi-
ion, we obse ed a loss o he p oli e a ion ma ke KI67 and a
sha p dec ease in B dU inco po a ion in auxin- ea ed umou s,
consis en wi h he pheno ype o educed g ow h o cul u ed
KPC cells. Since MYC p omo es immune e asion in panc ea ic
umou s,12 35 51 we analysed immune cell in il a ion on RUVBL1
deple ion and obse ed a ampan inc ease in CD3- posi i e cells
wi hin umou s on auxin ea men ( igu e 7A, online supple-
men al igu e S7A). We concluded ha RUVBL1 deple ion in
panc ea ic umou s induces immune cell in il a ion and he e-
o e phenocopies he gene ic silencing o MYC in simila umou
models.
Human panc ea ic umou s usually con ain ew immune cells,
which is hough o explain hei low esponse o immune check-
poin blockade. We he e o e combined auxin- media ed deple-
ion o RUVBL1 wi h adminis a ion o an αPD- 1 an ibody.
S ikingly, combina o ial ea men , bu no αPD- 1 ea men
alone, induced long- e m su i al o ou o he 11 mice e en
a e he ea men was e mina ed ( igu e 7B). We concluded
ha deple ion o RUVBL1 in panc ea ic cance leads o immune
in il a ion and induces umou sensi i i y o immune check-
poin blockade.
copy igh . on Sep embe 9, 2024 a Uni e si a sbiblio hek Zei sch i enab eilung. P o ec ed byh p://gu .bmj.com/Gu : i s published as 10.1136/gu jnl-2023-331519 on 31 May 2024. Downloaded om
1524 Vog M, e al. Gu 2024;73:1509–1528. doi:10.1136/gu jnl-2023-331519
Panc eas
and HDR empla e we e co ans ec ed in o KPC cells wi h Lipo-
ec amine 2000 (The mo Fishe Scien i ic). A e 72 hou s, cells
we e selec ed wi h 15 µg/mL blas icidin (In i oGen) o 10 days.
Colonies om single- cell clones we e ans e ed o 24- well
pla es and geno yped by PCR. PCR p oduc s we e pu i ied using
he GeneJET Gel Ex ac ion ki (The mo Fishe Scien i ic) and
sen o Sange sequencing (LGC Genomics). The used KPCAID-
RUVBL1 clone (C6) is a hemizygous clone in which he AID ag
was success ully in eg a ed in o one Ru bl1 allele, while in he
second allele dele ion o he s a codon ab oga ed exp ession
(see online supplemen al igu e 3B,C).
CRISPR knock- in o he AID sequence was also done a he
MYC locus o human cells as desc ibed abo e, wi h he ollowing
modi ica ions: HAs lanked he MYC s op codon and, a e PCR,
we e cloned in o pJET lanking he AID- V5- P2A- Blas casse e.
The sgRNA a ge ed he egion a ound he MYC s op codon,
and his plasmid and he HDR empla e we e co ans ec ed in o
A375 human melanoma cells.
Fo TIR1F74G exp ession in KPCAID- Ru bl1 cells, TIR1F74G was
cloned in o pRRLSin.cPPT.SFFV- IRES- Hyg o.WPRE. KPCAID-
Ru bl1 cells we e len i i ally ansduced wi h he ec o and
selec ed wi h 500 µg/mL hyg omycin (In i oGen) o 7 days.
AID- agged p o ein deg ada ion was induced by ea men wi h
1 µM auxin (30- 003- 10, BioAcademia), i no s a ed o he wise.
The auxin ehicle, DMSO, was used in expe imen s as he nega-
i e con ol. Fi e ly luci e ase was cloned in o he pRRLSin.
cPPT.SFFV- IRES- Pu o.WPRE backbone (we used pRRLSin.
cPPT.SFFV- IRES- Hyg o.WPRE wi h he hyg omycin esis ance
sequence exchanged o pu omycin). KPCAID- Ru bl1; TIR1 cells we e
in ec ed wi h len i i al supe na an and selec ed wi h 2 µg/mL
pu omycin (In i oGen) o 72 hou s.
SLAM-seq
Cells we e ea ed wi h auxin o 3 o 15 hou s and subsequen ly
wi h 800 µM 4sU (Sigma- Ald ich) o 2 hou s. Al e na i ely,
hey we e ea ed wi h 1 µM CB- 6644 (MedChemExp ess) o
20 hou s, ollowed by 200 nM OHT (H7904, Sigma- Ald ich)
o 4 hou s and 400 µM 4sU o 2 hou s. Cells we e ha es ed
in QIAzol lysis eagen (Qiagen), and RNA was ex ac ed by
phenol/chlo o o m/isoamyl alcohol (Ca l Ro h) ex ac ion and
p ecipi a ion wi h isop opanol (Ca l Ro h). Inco po a ed 4sU
was alkyla ed using 10 mM iodoace amide (The mo Fishe
Scien i ic), and he eac ion was quenched wi h 1 M DTT
(The mo Fishe Scien i ic). Alkyla ed RNA was pu i ied on
MinElu e columns (Qiagen). RNA in eg i y was e i ied using
he S anda d Sensi i i y RNA ki (Agilen Technologies) on a
F agmen Analyze sys em (Agilen Technologies). Samples ha
passed quali y checks we e used o lib a y p epa a ion using
he Quan Seq Fwd ki (Lexogen) o 15 cycles. They we e
sequenced o 75 cycles on a Nex Seq500 o o 120 cycles on a
Nex Seq2000 sequence (Illumina).
We used he GRAND- SLAM pipeline (V.2.0.7) o p ocess bo h
SLAM- seq da a se s. B ie ly, 10 n (6 n unique molecula iden i-
ie (UMI)+4 n space ) we e immed om he 5' ends o eads
(Fas qFil e p og am om he GRAND- SLAM pipeline), and
he sequencing adap e ( AGAT CGGA AGAG CACA CGTC TGAA
CTCC AGTCA) was immed om he 3' end using Cu adap
V.3.4. Nex , Bow ie 2 (V.2.3.0) wi h de aul pa ame e s was
used o disca d eads mapping o RNA (GenBank iden i ie
U13369.1) and o e i y he absence o Mycoplasma con amina-
ion. STAR V.2.5.3a was used o map all emaining eads wi h a
leng h o a leas 18 n agains a combined index o he mu ine
genome (Ensembl 102) and ERCC92 spike- ins (pa ame e s:
--ou Fil e Misma chNmax 20, --ou Fil e Sco eMinO e L ead
0.4, --ou Fil e Ma chNminO e L ead 0.4, --alignEndsType
Ex end5pO Reads12, --ou SAMa ibu es nM MD NH). Finally,
all eads mapping o he same genomic loca ion sha ing he same
UMI we e collapsed, and only misma ches ha occu ed in he
majo i y o hese eads we e e ained (DedupUMI p og am o
he GRAND- SLAM pipeline). The GRAND- SLAM p og am
was un wi h pa ame e – im 15 agains a combined index o
mu ine mRNAs (Ensembl 102) and he ERCC92 spike- ins, o
coun eads and o es ima e he new- o- o al RNA a io o each
sample and each mRNA.
The g andR package ( e sion 0.1.11 o he CB- 6644 da a se ,
and e sion 0.1.23 o he auxin da a se ) was used o quali y
con ol and downs eam analyses. The absence o cellula oxici y
o 4sU was con i med using he ‘Plo Toxici yTes RankAll’ unc-
ion. Genes we e il e ed o ha e a leas 50 eads in a leas
hal o he samples. Read coun s pe sample we e no malised by
di iding by he size ac o s (es ima eSizeFac o sFo Ma ix om
he DESeq2 R package) compu ed om ei he he o al numbe
o ERCC92 mapped eads o he o al numbe o mu ine mRNA
mapped eads. G ea e wi hin- eplica e a iabili y a e ERCC92
no malisa ion indica ed ha he a iance in ERCC spike- ins was
g ea e han in o al RNA con en o he samples. The e o e,
we con inued wi h mu ine mRNA size ac o - no malised coun s.
P incipal componen analysis showed an ex eme ou lie ( he
hi d eplica e o he 15- hou ime poin in he auxin da a se ),
which was he e o e excluded om u he analyses. P alues
we e compu ed on o al o new RNA using he Wald es imple-
men ed in DESeq2, and old changes we e es ima ed using he
PsiLFC es ima o om he l c package.
RNA-seq
Cells we e ea ed wi h 1 µM CB- 6644 o 24 hou s. RNA was
ex ac ed using he miRNeasy ki (Qiagen). RNA in eg i y was
e i ied using he S anda d Sensi i i y RNA ki (Agilen Technol-
ogies) on a F agmen Analyze sys em (Agilen Technologies).
Samples ha passed quali y checks we e p ocessed by deple ing
RNA wi h he NEBNex RNA deple ion ki 2 (NEB) and
subsequen lib a y p epa a ion using he NEBNex Ul a II Di ec-
ional RNA Lib a y P ep ki (NEB). Lib a ies we e sequenced
o 2×60 cycles on a Nex Seq2000 pla o m (Illumina). FASTQ
iles we e aligned o he mm39 genome using STAR V.2.5.3a.
Gene- le el eads (Ensembl e sion 111) we e coun ed using he
GenomicAlignmen s R package ( e sion 1.38.2) and di e en ial
gene exp ession analysis was pe o med wi h edgeR (V.4.0.16).
Ch oma in immunop ecipi a ion
Fo each immunop ecipi a ion sample, 50 million cells we e
c osslinked wi h o maldehyde ( inal concen a ion, 1%) o 10
min a oom empe a u e, as desc ibed. Glycine was added o
a inal concen a ion o 125 mM o s op he c osslinking, and
samples we e incuba ed o 5 min a oom empe a u e. Cells
we e washed wice wi h ice- cold PBS and esuspended in PBS
supplemen ed wi h p o ease and phospha ase inhibi o s (phos-
pha ase inhibi o cock ail 2/3, P5726, P0044; p o ease inhib-
i o cock ail, P8340, Sigma- Ald ich). Bu e s used in u he
s eps we e eshly supplemen ed wi h p o ease and phospha ase
inhibi o s.
To con ol o o e all changes in ch oma in binding, human
o mouse cell ch oma in was added o samples o KPC o A375
cells, espec i ely. In pa icula , 3 million U2OS os eosa coma
cells o NIH3T3 ib oblas s (6% o he s a ing cell numbe )
we e added o each sample. Then, samples we e lysed in lysis
copy igh . on Sep embe 9, 2024 a Uni e si a sbiblio hek Zei sch i enab eilung. P o ec ed byh p://gu .bmj.com/Gu : i s published as 10.1136/gu jnl-2023-331519 on 31 May 2024. Downloaded om

1525
Vog M, e al. Gu 2024;73:1509–1528. doi:10.1136/gu jnl-2023-331519
Panc eas
bu e I (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP- 40) a
4°C o 20 min. Nuclei we e collec ed by cen i uga ion (1500
pm o 15 min a 4°C), and he pelle s we e dissol ed in lysis
bu e II (10 mM T is pH 7.5, 150 mM NaCl, 1 mM EDTA,
1% NP- 40, 1% sodium deoxychola e, 0.1% SDS). C osslinked
ch oma in was agmen ed by sonica ion ( o al du a ion 16 min
wi h 10 s pulses and 45 s pauses). A agmen size dis ibu ion
o 150–300 bp was e i ied by aga ose gel elec opho esis. The
samples we e cen i uged (20 min a 14 000 pm a 4°C), and
he supe na an was aken as he shea ed ch oma in inpu o
immunop ecipi a ion.
Fo immunop ecipi a ion, 100 µL Dynabeads P o ein A and
P o ein G (The mo Fishe Scien i ic) we e p eincuba ed o e -
nigh wi h 10 μg p ima y an ibody in 5 g/L BSA in PBS. The an i-
bodies we e agains RUVBL1 (Cell Signaling, 74775) and MYC
(Abcam, ab32072). IgG (I5381, Sigma- Ald ich) was used as an
iso ype con ol. The an ibody- coupled beads we e washed h ee
imes wi h 5 g/L BSA in PBS. Shea ed ch oma in co esponding
o 50 million cells was added and incuba ed wi h o a ing o
6 hou s a 4°C. Then, he beads we e washed h ice wi h washing
bu e I (20 mM T is pH 8.1, 150 mM NaCl, 2 mM EDTA,
1% T i on X- 100, 0.1% SDS), washing bu e II (20 mM T is
pH 8.1, 500 mM NaCl, 2 mM EDTA, 1% T i on X- 100, 0.1%
SDS), washing bu e III (10 mM T is pH 8.1, 250 mM LiCl, 1
mM EDTA, 1% NP- 40, 1% sodium deoxychola e; including a
5 min incuba ion s ep wi h o a ion) and once wi h TE bu e
(The mo Fishe Scien i ic). Ch oma in–p o ein complexes we e
elu ed wice om he beads by incuba ing wi h 150 µL eshly
p epa ed elu ion bu e (100 mM NaHCO3, 1% SDS) o 15 min
a oom empe a u e, wi h o a ion. Dec osslinking o he elu ed
and inpu samples was done o e nigh , ollowed by diges ion
wi h p o einase K (Ca l Ro h) and RNase A ( inal concen a-
ion, 60 µg/mL). The DNA was pu i ied by phenol- chlo o o m
ex ac ion and p ecipi a ed wi h e hanol. The esul ing ChIP
DNA pelle s we e dissol ed in wa e o analysis.
ChIP-qPCR and ChIP-seq
To assess he e iciency o immunop ecipi a ion, ChIP DNA
pelle s we e analysed by qPCR on a S epOnePlus Real- Time PCR
Sys em (The mo Fishe Scien i ic) using he SYBR G een Mas e
Mix (The mo Fishe Scien i ic). Equal amoun s o ChIP DNA
and SYBR G een Mas e Mix we e added along wi h 0.5 μM
p ime s. qPCR assays we e done in echnical iplica es.
Fo ChIP- seq, qPCR- e i ied ChIP DNA was quan i ied using
he Quan - iT PicoG een dsDNA assay (The mo Fishe Scien-
i ic). Lib a y p epa a ion was done using he NEBNex Ul a
II DNA Lib a y P ep Ki o Illumina (NEB). The lib a ies we e
ampli ied using 13 PCR cycles. The concen a ion and size dis i-
bu ion o he lib a y we e e alua ed on a F agmen Analyze
(Agilen Technologies) using he NGS F agmen High Sensi i i y
Analysis Ki (1–6000 bp; Agilen Technologies). The lib a ies
we e sequenced on a Nex Seq500 pla o m o 75 cycles o a
Nex Seq2000 sequence o 120 cycles (Illumina).
FASTQ iles o inpu samples om auxin and DMSO- ea ed
cells we e combined. FASTQ iles we e aligned o he mm10
and hg19 genomes using Bow ie 2 V.2.3.4.1. BAM iles we e
no malised by a scaling ac o de e mined om he numbe o
human o mouse eads pe da a se , espec i ely. No malised
BAM iles we e so ed using SAM ools e sion 1.7 and con e ed
in o bedg aphs wi h bed ools e sion 2.26.0. Co e age in
p omo e egions ( ansc ip ion s a si e (TSS)±3 kb) was calcu-
la ed using bed ools co e age on all anno a ed Ensembl genes
( elease 102). Fo MYC and RUVBL1, ChIP- seq peaks we e
called using MACS2 e sion 2.2.7.1 wi h p alue cu - o s o
0.01 and 0.001, espec i ely. P omo e peaks we e de ined as
peaks o e lapping wi h p omo e s (TSS±3 kb). O e lap be ween
MYC and RUVBL1 peaks and genomic ea u e anno a ions we e
calcula ed using ChIPpeakAnno e sion 3.30.1 and compa ed
wi h he dis ibu ion o 1 million andom 300 bp in e als. To
plo hea maps, BAM iles we e con e ed o bigWig iles using
deepTools e sion 3.5.1, and a ead ma ix was calcula ed om
bigWig iles a ound he peak posi ions o RUVBL1, MYC and
he sha ed si es.
O e exp ession o MYC-ER and RUVBL1 mu an s
To o e exp ess exogenous MYC, KPC cells we e ansduced
ia len i i al in eg a ion wi h pRRLSin.cPPT.SFFV- IRES- Pu o.
WPRE con aining a MYC- ER inse . To o e exp ess RUVBL1,
KPC cells we e ansduced wi h pRRLSin.cPPT.SFFV- IRES- Pu o.
WPRE con aining a RUVBL1WT, RUVBL1A62T, RUVBL1D302N,
RUVBL1A62T, D302N, RUVBL1Δ94- 118, RUVBL1Δ102- 107 o
RUVBL1K108A open eading ame ia len i i al in eg a ion.
RUVBL1 mu an s we e o e exp essed in KPC o KPCAID- Ru bl1;
TIR1 cells by len i i al ansduc ion wi h pRRLSin.cPPT.SFFV-
IRES- Pu o.WPRE, espec i ely. Cells we e selec ed wi h 2 µg/
mL pu omycin (In i oGen) o 3 days.
E. coli
exp ession and pu i ica ion o MYC1-163 and RUVBL1/2
Human MYC (1- 163) was cloned in o a modi ied pET28b
ec o ollowing an N- e minal Hisx6- Mal ose binding p o ein
(MBP) ag and a obacco e ch i us (TEV) p o ease clea age si e
(Addgene: 29654). P o ein was exp essed in he Bl21 (DE3)
RIL E. coli s ain a 37°C. P o ein exp ession was induced by
adding 0.5 mM IPTG o he medium when he cul u e eached
an op ical densi y o 0.5 and le ing he cul u e g ow o an addi-
ional 3 hou s a 37°C. P o ein pu i ica ion s eps we e pe o med
a 4°C.
The cells we e collec ed by cen i uga ion and esuspended in
lysis bu e (20 mM T is- HCl pH 7.9, 30 mM imidazole, 500 mM
NaCl, 10% glyce ol, 5 mM be a- me cap oe hanol, 0.284 µg/mL
leupep in, 1.37 µg/mL peps a in A, 0.17 mg/mL PMSF, 0.33 mg/
mL benzamidine). Cells we e lysed by sonica ion, and lysa es
we e cla i ied by cen i uga ion. Cla i ied lysa e was applied o
a 5 mL HisT ap column equilib a ed in lysis bu e . The column
was washed wi h lysis bu e and addi ionally wi h i e column
olumes o a high sal bu e (lysis bu e wi h 1 M NaCl) be o e
e u ning o lysis bu e . P o ein was elu ed om he HisT ap
column wi h elu ion bu e (20 mM T is- HCl pH 7.9, 500 mM
imidazole pH 8.0, 500 mM NaCl, 10% glyce ol, 5 mM be a-
me cap oe hanol) and loaded di ec ly on o a 10 mL amylose
column (New England Biolabs). The amylose column was washed
wi h lysis bu e , and bound p o ein was elu ed wi h a mal ose-
con aining bu e (20 mM T is- HCl pH 7.9, 30 mM imidazole
pH 8.0, 500 mM NaCl, 116 mM mal ose, 10% glyce ol, 5 mM
be a- me cap oe hanol). F ac ions con aining MYC, iden i ied by
SDS- PAGE and Coomassie s aining, we e pooled and concen-
a ed on an Amicon Millipo e ul a il a ion de ice (10 000
molecula weigh cu - o ). Concen a ed p o ein was applied
o a HiLoad Supe dex 200 16/600pg column (GE Heal hca e)
equilib a ed in size exclusion bu e (20 mM T is- HCl pH 7.9,
500 mM NaCl, 10% glyce ol, 1 mM DTT). F ac ions con aining
MYC we e pooled and concen a ed again. P o ein concen a-
ion was de e mined using he calcula ed ex inc ion coe icien
o His6- MBP- MYC and he abso bance a 280 nm. P o ein was
aliquo ed, snap ozen in liquid ni ogen and s o ed a −80°C
un il use.
copy igh . on Sep embe 9, 2024 a Uni e si a sbiblio hek Zei sch i enab eilung. P o ec ed byh p://gu .bmj.com/Gu : i s published as 10.1136/gu jnl-2023-331519 on 31 May 2024. Downloaded om
1526 Vog M, e al. Gu 2024;73:1509–1528. doi:10.1136/gu jnl-2023-331519
Panc eas
Full- leng h human RUVBL1 and RUVBL2 we e cloned in o
ec o s 14B (Addgene: 48308) and 14A (Addgene: 48307),
espec i ely, ia liga ion- independen cloning. 14B con ains a
Hisx6 ag ollowed by a TEV clea age si e. The ec o s we e
combined o c ea e a co- exp ession ec o . The p o eins we e
exp essed ia au oinduc ion in BL21 (DE) RIL LOBSTR E. coli.
P o ein pu i ica ion s eps we e pe o med a 4°C unless o he -
wise no ed.
Cells we e collec ed by cen i uga ion and esuspended
in bu e A (20 mM T is- HCl pH 7.9, 30 mM imidazole pH
8.0, 300 mM NaCl, 1 mM MgCl2, 10% glyce ol, 5 mM be a-
me cap oe hanol, 0.284 µg/mL leupep in, 1.37 µg/mL peps a in
A, 0.17 mg/mL PMSF, 0.33 mg/mL benzamidine). Cells we e
lysed by sonica ion, and lysa es we e cla i ied by cen i uga ion.
Cla i ied lysa e was applied o a 5 mL HisT ap column equili-
b a ed in bu e A. The column was washed wi h bu e A and
wi h i e column olumes o a high sal bu e (lysis bu e as
abo e wi h 1 M NaCl) be o e e u ning o lysis bu e and hen
low sal bu e (lysis bu e wi h 150 mM NaCl). P o ein was
elu ed om he HisT ap column wi h elu ion bu e (20 mM
T is- HCl pH 7.9, 500 mM imidazole pH 8.0, 150 mM NaCl,
1 mM MgCl2, 10% glyce ol, 5 mM be a- me cap oe hanol) and
loaded di ec ly on o a 5 mL HiT ap Q column (GE Heal h-
ca e) equilib a ed in low sal bu e . The HiT ap Q column was
washed wi h low sal bu e , and bound p o eins we e elu ed
wi h a linea g adien (30 min, 1.5 mL/min) in o 100% high sal
bu e . F ac ions we e e alua ed by SDS- PAGE and Coomassie
s aining, and hose con aining RUVBL1/2 we e pooled and
mixed wi h 1.5 mg His6- TEV p o ease. The p o ein was dial-
ysed agains 1 L lysis bu e o e nigh and hen applied o a 5 mL
HisT ap column equilib a ed in lysis bu e o emo e unclea ed
p o ein and TEV p o ease. The ollow- h ough was collec ed
and concen a ed wi h an Amicon Millipo e ul a il a ion de ice
(30 000 molecula weigh cu - o ). The concen a ed p o ein was
applied o a Supe ose 6 Inc ease 10/300 column (GE Heal h-
ca e) equilib a ed in size exclusion bu e (20 mM T is- HCl pH
7.9, 200 mM NaCl, 1 mM MgCl2, 10% glyce ol, 5 mM be a-
me cap oe hanol). Elu ed p o ein was iden i ied by SDS- PAGE
and Coomassie s aining, and ac ions con aining RUVBL1 and
RUVBL2 we e pooled and concen a ed again. P o ein concen-
a ion was de e mined using he calcula ed ex inc ion coe i-
cien o RUVBL1/2 and he abso bance a 280 nm. P o ein was
aliquo ed, snap ozen in liquid ni ogen and s o ed a −80°C
un il use.
G adien cen i uga ion
Fo he complex o ma ion assay, pu i ied RUVBL1/2 (20 µM),
MYC (20 µM) o bo h we e mixed wi h ADP- BeF (1 mM) in
20 mM Na•HEPES pH 7.4, 50 mM NaCl, 1 mM DTT, 3 mM
MgCl2. The solu ion (100 µL) was applied o a 10–30% suc ose
g adien and cen i uged o 16 hou s a 32 000 pm a 4°C in an
SW41 o o (Beckman). Then, 200 µL samples we e sequen ially
ac iona ed om he op o he g adien . 15 µL o each sample
was analysed by 10% SDS- PAGE, and p o eins we e iden i ied
by Coomassie blue s aining.
Pull-down assay
His6- MBP- MYC1- 163 (5 µM) was incuba ed wi h 15 µM o he
ull- leng h RUVBL1/2 complex in a inal assay bu e con aining
50 mM NaCl, 20 mM T is- HCl pH 7.9, 3 mM MgCl2, 1 mM
DTT and 10% glyce ol ( inal olume 10 µL). The p o ein was
hen added o 50 µL o amylose beads (New England Biolabs)
ha we e equilib a ed in he assay bu e . The complexes we e
incuba ed o 20 min a oom empe a u e in a he momixe
(300 pm). The beads we e hen washed h ee imes wi h 500
µL assay bu e . A e he inal wash, p o ein was elu ed om
he beads wi h 30 µL o assay bu e supplemen ed wi h 116 mM
mal ose. The elu ed p o ein was applied o a 10% SDS- PAGE
and p o eins we e isualised using Coomassie blue.
Immunohis ochemis y
A e explan a ion, umou s we e ixed in 4% o maldehyde
o e nigh and washed wi h 70% e hanol. They we e dehy-
d a ed by subsequen imme sing in inc easing concen a ions o
e hanol and inally xylol (Ca l Ro h). Tumou s we e embedded
in pa a in, and issue sec ions we e cu and placed on slides.
Sec ions we e depa a inised by imme sing in xylol and ehy-
d a ed by imme sing in dec easing concen a ions o e hanol.
An igens we e e ie ed by boiling he slides in 10 mM sodium
ci a e bu e pH 6 o 15 min. Pe oxidases we e blocked by
ea ing o 10 min wi h 3% H2O2. Sec ions we e washed wice
in TBS and blocked o 1 hou a oom empe a u e wi h 10%
goa se um (G6767, Sigma- Ald ich) in TBS. Sec ions we e incu-
ba ed wi h a p ima y an ibody (RUVBL1, Cell Signaling, 74775S,
1:100; KI67, RM- 9106- S, 1:200; B dU, Bio- Rad, OBT0030G,
1:200; CD3, P o ein ech, 17617- 1- AP, 1:10 000) in 5% goa
se um in TBS o e nigh a 4°C. Slides we e washed h ice wi h
TBS, once again blocked in 10% goa se um in TBS, and incu-
ba ed wi h an HRP- conjuga ed seconda y an ibody (an i- abbi
IgG: The mo Fishe Scien i ic, B40962; an i- a IgG: Sigma-
Ald ich, GENA935) o 1 hou a oom empe a u e. Slides
we e washed h ice wi h TBS and signals we e de eloped using
he SignalS ain DAB Subs a e ki (8059, Cell Signaling). Slides
we e coun e s ained wi h haema oxylin solu ion (GHS332,
Sigma- Ald ich) and dehyd a ed in inc easing concen a ions o
e hanol and inally xylol be o e moun ing hem wi h Cy oseal
60 moun ing medium (The mo Fishe Scien i ic) and le ing
hem d y. Slides we e scanned using a Panno amic Desk slide
scanne (3DHISTECH), and images we e analysed using QuPa h
so wa e V.0.3.2. Fo he CD3 s aining o umou s induced by
24031 o 9172 cells ea ed wi h CB- 6644 o ehicle, simila
size (small) umou lesions (24031: 8200–12 000 cells, 9172:
380–1600 cells) we e compa ed. The human TMA ep esen ing
p ima y PDAC issue and umou s oma om 31 indi iduals
and benign panc ea ic issue, ha is, acina and duc al issue,
om 24 indi iduals who unde wen su ge y o PDAC a he
uni e si y hospi al in Wü zbu g, Ge many be ween 2010 and
2020 was s ained o RUVBL1 as desc ibed abo e. His osco e
dis ibu ions we e compa ed wi h he Wilcoxon signed- ank es .
Liquid ch oma og aphy–MS quan i ica ion o CB-6644 om
issue
Fo he analysis o CB- 6644 in issues, samples we e homo-
genised in 19 olumes o me hanol/wa e (80/20, / ) in Eppen-
do ubes using a po e el ehjem homogenisa o equipped wi h
a s ainless s eel pis il (10 s okes a 1200 pm). 200 µL o he
esul ing homogena e o , in case o blood samples, 10 µL sample
in 200 µL me hanol/wa e (80/20, / ) was dilu ed wi h 628 µL
o 0.01 µM lami udine in me hanol/wa e (80/20, / ), cen i-
uged (2 min maximum pm) and he esul ing supe na an was
applied o ac i a ed (wi h 280 µL ace oni ile) and equilib a ed
(wi h 280 µL me hanol/H2O (80/20, / )) C18- SPE columns
(Phenomenex S a a C18- E (50 mg) (Ascha enbu g, Ge many)).
Ano he 180 µL e hanol/H2O (80/20, / ) was applied o he
column and he elua es we e collec ed in an Eppendo ube and
e apo a ed o d yness in a o a y acuum concen a o (speed
copy igh . on Sep embe 9, 2024 a Uni e si a sbiblio hek Zei sch i enab eilung. P o ec ed byh p://gu .bmj.com/Gu : i s published as 10.1136/gu jnl-2023-331519 on 31 May 2024. Downloaded om
1527
Vog M, e al. Gu 2024;73:1509–1528. doi:10.1136/gu jnl-2023-331519
Panc eas
ac). P io analysis, he d y esidues we e edissol ed in 75 µL
o 5 mM NH4OAc in ace oni ile/H2O (50/50, / ). Following
cen i uga ion o 2 min a maximum pm, he supe na an was
ans e ed on o au osample glass ials and s o ed a 15°C o
u he analysis.
Liquid ch oma og aphy (LC)–MS analysis was pe o med
using a Q Exac i e mass spec ome e coupled o a Dionex
U3000 UHPLC sys em (The mo Fishe Scien i ic). The mass
spec ome e was ope a ed in ull MS posi i e mode applying he
ollowing MS pa ame e s: scan ange, 69–1000 m/z; esolu ion,
70 000; au oma ic gain con ol a ge , 3E6; maximum injec ion
ime, 200 ms; shea h gas, 30; auxilia y gas, 10; sweep gas, 3;
sp ay ol age, 3.6 kV; capilla y empe a u e, 320°C; S- lens adio
equency le el, 55.0; auxilia y gas hea e empe a u e, 120°C.
The LC sys em was i ed wi h an Accuco e Biphenyl column
(2.6 μm pa icles, 100×2.1 mm) (The mo Scien i ic, B emen,
Ge many) and pa icle il e (Supelco ColumnSa e 0.5 µm
(Me ck, Da ms ad , Ge many; 55214- U)). The column empe a-
u e was main ained a 45°C. The mobile phase was composed
o 5 mM NH4OAc in ace oni ile/H2O (5/95, / ) (sol en A)
and 5 mM NH4OAc in ace oni ile/H2O (95/5, / ) (sol en B).
Compounds we e elu ed a a low a e o 0.2 mL/min, applying
a g adien o 10% sol en B o 2 min, ollowed by a linea
dec ease o 100% sol en B wi hin 8 min, hen main aining
100% sol en B o 9 min, hen e u ning o 10% sol en B in 1
min, and 5 min 10% sol en B o column equilib a ion be o e
each injec ion.
Anno a ion and da a e alua ion: peaks co esponding o he
calcula ed monoiso opic masses (MIM±2 mMU) we e in eg a ed
using T aceFinde so wa e (V.3.3.350.0; The mo Fishe Scien-
i ic). Absolu e quan i ica ion o compounds was pe o med by
in e pola ion o he co esponding s anda d cu es ob ained
om comme cially a ailable compounds unning wi h he same
ba ch o samples.
Au ho a ilia ions
1Cance Sys ems Biology G oup, Chai o Biochemis y and Molecula Biology,
Theodo Bo e i Ins i u e, Uni e si y o Wü zbu g, Wü zbu g, Ge many
2Ins i u e o Biochemis y, Uni e si y o Kiel, Kiel, Ge many
3Depa men o Biology, Massachuse s Ins i u e o Technology, Camb idge,
Massachuse s, USA
4Ins i u e o Pha maceu ical Chemis y, Goe he- Uni e si y F ank u , F ank u am
Main, Ge many
5Max Planck Resea ch G oup and Ins i u e o Sys ems Immunology, Uni e si y o
Wü zbu g, Wü zbu g, Ge many
6Chai o Biochemis y and Molecula Biology, Theodo Bo e i Ins i u e, Uni e si y o
Wü zbu g, Wü zbu g, Ge many
7Depa men o Gene al, Visce al, T ansplan a ion, Vascula and Pedia ic Su ge y,
Uni e si y Hospi al Wü zbu g, Wü zbu g, Ge many
8Comp ehensi e Pneumology Cen e (CPC)/Ins i u e o Lung Heal h and Immuni y
(LHI), Helmhol z Munich, Membe o he Ge man Cen e o Lung Resea ch (DZL/
CPC- M), Munich, Ge many
9Ludwig- Maximilian- Uni e si ä München (LMU), Munich, Ge many
10Ins i u e o T ansla ional Cance Resea ch, TUM School o Medicine and Heal h,
Munich, Ge many
11Ins i u e o Pa hology, Uni e si y o Wü zbu g, Wü zbu g, Ge many
12Compu a ional Sys ems Vi ology and Bioin o ma ics, Ins i u e o Vi ology and
Immunobiology, Uni e si y o Wü zbu g, Wü zbu g, Ge many
X Ma kus Die enbache @die enbache l
Acknowledgemen s We hank And é Ku schke and Sa ah Hess o excellen
echnical suppo . Vale ie Ma a ese p o ided excellen scien i ic edi ing. SK and AH
a e g a e ul o suppo by he S uc u al Genomics Conso ium (SGC), a egis e ed
cha i y (1097737) ha ecei es unds om Baye , Boeh inge Ingelheim, B is ol
Meye Squibb, Genen ech, Genome Canada h ough On a io Genomics Ins i u e
(OGI- 196), EU/EFPIA/OICR/McGill/KTH/Diamond Inno a i e Medicines Ini ia i e 2
Join Unde aking (EUbOPEN g an 875510), Janssen, P ize and Takeda. SK is also
unded by he Ge man Cance Resea ch Cen e (DKTK), he F ank u Cance Ins i u e
(FCI) and he BMBF Clus e o he u u e p og amme PROXIDRUGS.
Con ibu o s MV and NDS conduc ed and in e p e ed mos expe imen s wi h
help om all au ho s. NDS pe o med all in i o expe imen s. YR, AG- W, GG, DS
and ME suppo ed he in i o expe imen s. YCG p oduced he MYC- AID cell line
and helped wi h cell iabili y expe imen s. JH pe o med ChIP- seq expe imen s. LG
and BM pe o med RUVBL1 ChIP- qPCR assays. BA pe o med and in e p e ed low
cy ome y expe imen s. KS, FK, TH and SMV pe o med and in e p e ed RUVBL1- MYC
in e ac ion expe imen s. SK and AH syn hesised compounds o he AID sys em. FE
p o ided suppo o he SLAM- seq expe imen . EW concep ualised he p ojec and
designed and supe ised he expe imen s. EW, MV and NDS w o e he manusc ip
wi h inpu om all au ho s. EW is he gua an o o he s udy who accep s ull
esponsibili y o he wo k and he conduc o he s udy, had access o he da a and
con olled he decision o publish.
Funding This wo k was suppo ed by g an s om he Ge man Resea ch Founda ion
(DFG, WO 2108/2- 1 and GRK 2243 o EW), he Ge man Cance Aid ( unding o
TACTIC o SK and EW) and he Eu opean Resea ch Council (Ta MYC and PROTAC-
PDAC o EW).
Compe ing in e es s None decla ed.
Pa ien and public in ol emen Pa ien s and/o he public we e no in ol ed in
he design, o conduc , o epo ing, o dissemina ion plans o his esea ch.
Pa ien consen o publica ion No applicable.
E hics app o al Animal p ocedu es we e app o ed by he Dis ic Go e nmen o
Lowe F anconia unde he p o ocol numbe s RUF- 55.2.2- 2532- 2- 1372 and RUF-
55.2- 2532- 2- 148 and conduc ed acco ding o he Ge man Animal P o ec ion Law
§8 Abs. 1 Tie SchG. All human samples a e comple ely anonymised, and hei use
has been app o ed by he local e hics commi ee (E hics Commi ee o Uni e si y o
Wü zbu g, app o al numbe 20230612 02).
P o enance and pee e iew No commissioned; ex e nally pee e iewed.
Da a a ailabili y s a emen Da a a e a ailable in a public, open access
eposi o y. P ima y sequencing da a (shRNA sc een, SLAM- seq, ChIP- seq, RNA- seq)
ha e been deposi ed in he Gene Exp ession Omnibus unde he accession numbe
GSE216095. All o he da a ele an o he s udy a e included in he a icle o
uploaded as supplemen a y in o ma ion.
Supplemen al ma e ial This con en has been supplied by he au ho (s). I
has no been e ed by BMJ Publishing G oup Limi ed (BMJ) and may no ha e
been pee - e iewed. Any opinions o ecommenda ions discussed a e solely hose
o he au ho (s) and a e no endo sed by BMJ. BMJ disclaims all liabili y and
esponsibili y a ising om any eliance placed on he con en . Whe e he con en
includes any ansla ed ma e ial, BMJ does no wa an he accu acy and eliabili y
o he ansla ions (including bu no limi ed o local egula ions, clinical guidelines,
e minology, d ug names and d ug dosages), and is no esponsible o any e o
and/o omissions a ising om ansla ion and adap a ion o o he wise.
Open access This is an open access a icle dis ibu ed in acco dance wi h he
C ea i e Commons A ibu ion 4.0 Unpo ed (CC BY 4.0) license, which pe mi s
o he s o copy, edis ibu e, emix, ans o m and build upon his wo k o any
pu pose, p o ided he o iginal wo k is p ope ly ci ed, a link o he licence is gi en,
and indica ion o whe he changes we e made. See:h ps://c ea i ecommons.o g/
licenses/by/4.0/.
ORCID iDs
Ma kusVog h p://o cid.o g/0000-0002-4525-2901
Ne enkaDud a ski S anko ic h p://o cid.o g/0000-0002-3857-1760
Ka ha inaSchneide h p://o cid.o g/0009-0009-4594-1609
A minWiege ing h p://o cid.o g/0000-0001-7777-0909
Ma kusDie enbache h p://o cid.o g/0000-0002-7402-7949
Die e Sau h p://o cid.o g/0000-0001-5874-0210
Elma Wol h p://o cid.o g/0000-0002-5299-6335
REFERENCES
1 Pich O, Bailey C, Wa kins TBK, e al. The ansla ional challenges o p ecision oncology.
Cance Cell 2022;40:458–78.
2 Luo G, Jin K, Cheng H, e al. P ognosis o dis al panc ea ic cance s con olled by s age.
Exp The Med 2020;20:1091–7.
3 Ne ala- Plagemann C, Hidalgo M, Ga ido- Laguna I. F om s a e- o - he- a ea men s
o no el he apies o ad anced- s age panc ea ic cance . Na Re Clin Oncol
2020;17:108–23.
4 Bea AS, Vonde heide RH, O’Ha a MH. Challenges and oppo uni ies o panc ea ic
cance immuno he apy. Cance Cell 2020;38:788–802.
5 Ca oll PA, F eie BW, Ma hsya aja H, e al. The MYC ansc ip ion ac o ne wo k:
balancing me abolism, p oli e a ion and oncogenesis. F on Med 2018;12:412–25.
6 Dang CV. MYC on he pa h o cance . Cell 2012;149:22–35.
7 Schaub FX, Dhankani V, Be ge AC, e al. Pan- cance al e a ions o he MYC oncogene
and i s p oximal ne wo k ac oss he cance genome A las. Cell Sys 2018;6:282–300.
copy igh . on Sep embe 9, 2024 a Uni e si a sbiblio hek Zei sch i enab eilung. P o ec ed byh p://gu .bmj.com/Gu : i s published as 10.1136/gu jnl-2023-331519 on 31 May 2024. Downloaded om
1528 Vog M, e al. Gu 2024;73:1509–1528. doi:10.1136/gu jnl-2023-331519
Panc eas
8 Adams JM, Ha is AW, Pinke CA, e al. The C- Myc oncogene d i en by
immunoglobulin enhance s induces lymphoid malignancy in ansgenic mice. Na u e
1985;318:533–8.
9 Mu phy DJ, Jun ila MR, Pouye L, e al. Dis inc h esholds go e n Myc’s biological
ou pu in i o. Cance Cell 2008;14:447–57.
10 Dhanaseka an R, Deu zmann A, Mahauad- Fe nandez WD, e al. The MYC Oncogene
- he g and O ches a o o cance g ow h and immune e asion. Na Re Clin Oncol
2022;19:23–36.
11 Jain M, A ani is C, Chu K, e al. Sus ained loss o a neoplas ic pheno ype by b ie
inac i a ion o MYC. Science 2002;297:102–4.
12 Sodi NM, Ko le e RM, Ba he VJA, e al. MYC ins uc s and main ains panc ea ic
adenoca cinoma pheno ype. Cance Disco 2020;10:588–607.
13 Ko le e RM, Sodi NM, Wilson CH, e al. Myc coope a es wi h Ras by p og amming
in lamma ion and immune supp ession. Cell 2017;171:1301–15.
14 Casey SC, Tong L, Li Y, e al. MYC egula es he an i umo immune esponse h ough
CD47 and PD- L1. Science 2016;352:227–31.
15 Soucek L, Whi ield J, Ma ins CP, e al. Modelling Myc inhibi ion as a cance he apy.
Na u e 2008;455:679–83.
16 Soucek L, Whi ield JR, Sodi NM, e al. Inhibi ion o Myc amily p o eins e adica es
k as- d i en lung cance in mice. Genes De 2013;27:504–13.
17 Beaulieu M- E, Jause T, Massó-Vallés D, e al. In insic cell- pene a ing ac i i y
p opels omomyc om p oo o concep o iable an i- MYC he apy. Sci T ansl Med
2019;11:eaa 5012.
18 Ga alda E, Beaulieu M- E, Mo eno V, e al. MYC a ge ing by OMO- 103 in solid
umo s: a phase 1 ial. Na Med 2024;30:762–71.
19 Adiseshaiah PP, C is RM, Hook SS, e al. Nanomedicine s a egies o o e come he
pa hophysiological ba ie s o Panc ea ic cance . Na Re Clin Oncol 2016;13:750–65.
20 Nai SK, Bu ley SK. X- ay s uc u es o Myc- Max and mad- Max ecognizing DNA.
molecula bases o egula ion by p o o- oncogenic ansc ip ion ac o s. Cell
2003;112:193–205.
21 Blackwood EM, Eisenman RN. Max: a helix- loop- helix zippe p o ein ha o ms a
sequence- speci ic DNA- binding complex wi h Myc. Science 1991;251:1211–7.
22 McMahon SB, Van Buski k HA, Dugan KA, e al. The no el ATM- ela ed p o ein TRRAP
is an essen ial co ac o o he C- Myc and E2F oncop o eins. Cell 1998;94:363–74.
23 Thomas LR, Adams CM, Wang J, e al. In e ac ion o he oncop o ein ansc ip ion
ac o MYC wi h i s ch oma in co ac o WDR5 is essen ial o umo main enance.
P oc Na l Acad Sci USA 2019;116:25260–8.
24 Lo enzin F, Bena y U, Baluapu i A, e al. Di e en p omo e a ini ies accoun o
speci ici y in MYC- dependen gene egula ion. Eli e 2016;5:e15161.
25 Ge lach JM, Fu e M, Gallan M, e al. PAF1 complex componen leo1 helps ec ui
d osophila MYC o p omo e s. P oc Na l Acad Sci U S A 2017;114:E9224–32.
26 End es T, Sol ie D, Heidelbe ge JB, e al. Ubiqui yla ion o MYC couples ansc ip ion
elonga ion wi h double- s and b eak epai a ac i e p omo e s. Mol Cell
2021;81:830–44.
27 F ank SR, Pa isi T, Taube S, e al. MYC ec ui s he TIP60 His one ace yl ans e ase
complex o Ch oma in. EMBO Repo s 2003;4:575–80.
28 Baluapu i A, Ho s e e J, Dud a ski S anko ic N, e al. MYC ec ui s Sp 5 o
RNA polyme ase II o p omo e p ocessi e ansc ip ion elonga ion. Mol Cell
2019;74:674–87.
29 Kalka M, Rese ca D, Lou enco C, e al. MYC p o ein in e ac ome p o iling e eals
unc ionally dis inc egions ha coope a e o d i e umo igenesis. Mol Cell
2018;72:836–48.
30 Dinga D, Tu WB, Rese ca D, e al. MYC dephospho yla ion by he Pp1/PNUTS
phospha ase complex egula es ch oma in binding and p o ein s abili y. Na Commun
2018;9:3502.
31 Büchel G, Ca s ensen A, Mak K- Y, e al. Associa ion wi h au o a- A con ols N- MYC-
dependen p omo e escape and pause elease o RNA polyme ase II du ing he cell
cycle. Cell Rep 2017;21:3483–97.
32 Fellmann C, Ho mann T, S idha V, e al. An Op imized mic oRNA backbone o
e ec i e single- copy RNAi. Cell Rep 2013;5:1704–13.
33 Vog M, Wol E. Gse216095. gene exp ession omnibus [da ase ].
34 Hingo ani SR, Wang L, Mul ani AS, e al. T p53R172H and K asg12D coope a e
o p omo e ch omosomal ins abili y and widely me as a ic Panc ea ic Duc al
adenoca cinoma in mice. Cance Cell 2005;7:469–83.
35 K enz B, Gebha d - Wol A, Ade CP, e al. MYC- and Miz1- dependen esicula
anspo o double- s and RNA con ols immune e asion in Panc ea ic Duc al
adenoca cinoma. Cance Res 2021;81:4242–56.
36 Ghandi M, Huang FW, Jané-Valbuena J, e al. Nex - gene a ion cha ac e iza ion o he
cance cell line encyclopedia. Na u e 2019;569:503–8.
37 Moullan N, Mouchi oud L, Wang X, e al. Te acyclines dis u b mi ochond ial unc ion
ac oss euka yo ic models: a call o cau ion in BIOMEDICAL esea ch. Cell Rep
2015;10:1681–91.
38 Sa agalla S, Kolapalli SP, Vallabhapu apu S. The wo sides o Yy1 in cance : a iend
and a oe. F on Oncol 2019;9:1230.
39 Fuchs M, Ge be J, D apkin R, e al. The P400 complex is an essen ial E1A
ans o ma ion a ge . Cell 2001;106:297–307.
40 Yu Z, Xu Q, Wang G, e al. DNA opoisome ase Iialpha and RAD21 cohesin complex
componen a e p edic ed as po en ial he apeu ic a ge s in bladde cance . Oncol Le
2019;18:518–28.
41 Auge A, Ma hsya aja H, Ib ahim AH, e al. MAX unc ions as a umo supp esso and
ewi es me abolism in small cell lung cance . Cance Cell 2020;38:97–114.
42 Soucek L, Nasi S, E an GI. Omomyc exp ession in skin p e en s Myc- induced
papilloma osis. Cell Dea h Di e 2004;11:1038–45.
43 Demma MJ, Mapelli C, Sun A, e al. Omomyc e eals new mechanisms o inhibi he
MYC oncogene. Mol Cell Biol 2019;39:e00248- 19.
44 Das SK, Kuzin V, Came on DP, e al. “MYC assembles and s imula es opoisome ases 1
and 2 in a " opoisome"” Mol Cell 2022;82:140–58.
45 Pe an I, Madha an S, Bye s SW, e al. Cu a ion o he panc ea ic duc al
adenoca cinoma subse o he cance genome A las is essen ial o accu a e
conclusions abou su i al- ela ed molecula mechanisms. Clin Cance Res
2018;24:3813–9.
46 Go ynia S, Bandei as TM, Pinho FG, e al. S uc u al and unc ional insigh s in o
a dodecame ic molecula machine - he Ru bl1/Ru bl2 complex. J S uc Biol
2011;176:279–91.
47 Wang H, Li B, Zuo L, e al. The ansc ip ional coac i a o Ru bl2 egula es Pol II
clus e ing wi h di e se ansc ip ion ac o s. Na Commun 2022;13:5703.
48 Yesbola o a A, Sai o Y, Ki amo o N, e al. The auxin- inducible deg on 2 echnology
p o ides sha p deg ada ion con ol in yeas , mammalian cells, and mice. Na Commun
2020;11:5701.
49 Assimon VA, Tang Y, Va gas JD, e al. CB- 6644 is a selec i e inhibi o o he Ru bl1/2
complex wi h an icance ac i i y. ACS Chem Biol 2019;14:236–44.
50 Muha M, Ebe A, Neumann T, e al. SLAM- Seq de ines di ec gene- egula o y
unc ions o he B d4- MYC axis. Science 2018;360:800–5.
51 Mu halagu N, Mon e e de T, Ra o- I aolagoi ia X, e al. Rep ession o he ype I
in e e on pa hway unde lies MYC- and KRAS- dependen e asion o NK and B cells in
panc ea ic duc al adenoca cinoma. Cance Disco 2020;10:872–87.
52 Bellos a P, Hul T, Balla Diop S, e al. MYC in e ac s gene ically wi h Tip48/Rep in and
Tip49/Pon in o con ol g ow h and p oli e a ion du ing D osophila de elopmen . P oc
Na l Acad Sci U S A 2005;102:11799–804.
53 Wood MA, McMahon SB, Cole MD. An ATPase/Helicase complex is an essen ial
co ac o o oncogenic ans o ma ion by C- Myc. Mol Cell 2000;5:321–30.
54 Li M, Yang L, Chan AKN, e al. Epigene ic con ol o ansla ion checkpoin and umo
p og ession ia Ru bl1- Ee 1A1 axis. Ad Sci (Weinh) 2023;10:2206584.
55 Be eshchenko O, Mancini E, Luciani L, e al. Pon in is essen ial o mu ine
hema opoie ic s em cell su i al. Haema ologica 2012;97:1291–4.
56 Zhang G, Li S, Cheng KW, e al. AAA A pases as he apeu ic a ge s: s uc u e,
unc ions, and small- molecule inhibi o s. Eu J Med Chem 2021;219:113446.
57 Siladi AJ, Wang J, Flo ian AC, e al. WIN si e inhibi ion dis up s a subse o WDR5
unc ion. Sci Rep 2022;12:1848.
58 Aho ER, Wang J, Goglio i RD, e al. Displacemen o WDR5 om ch oma in by a WIN
si e inhibi o wi h picomola a ini y. Cell Rep 2019;26:2916–28.
59 Thomas LR, Wang Q, G ieb BC, e al. In e ac ion wi h Wd 5 p omo es a ge gene
ecogni ion and umo igenesis by MYC. Mol Cell 2015;58:440–52.
60 de P e is S, K ess TR, Mo elli MJ, e al. In eg a i e analysis o RNA polyme ase
II and ansc ip ional dynamics upon MYC ac i a ion. Genome Res
2017;27:1658–64.
61 Ullman NA, Bu cha d PR, Dunne RF, e al. Immunologic s a egies in panc ea ic
cance : making cold umo s ho . J Clin Oncol 2022;40:2789–805.
62 Pe cie du Se N, Hu s V, Ahluwalia A, e al. The ARRIVE guidelines 2.0: upda ed
guidelines o epo ing animal esea ch. PLoS Biol 2020;18:e3000410.
63 Pelosso R, Fai child L, Huang C- H, e al. P edic ion o po en shRNAs wi h a
sequen ial classi ica ion algo i hm. Na Bio echnol 2017;35:350–3.
64 Nishimu a K, Fukagawa T, Takisawa H, e al. An auxin- based deg on sys em o he
apid deple ion o p o eins in nonplan cells. Na Me hods 2009;6:917–22.
copy igh . on Sep embe 9, 2024 a Uni e si a sbiblio hek Zei sch i enab eilung. P o ec ed byh p://gu .bmj.com/Gu : i s published as 10.1136/gu jnl-2023-331519 on 31 May 2024. Downloaded om