Ci a ion: Loxha, L.; Ib ahim, N.K.;
S asche, A.S.; Cina , B.; Dolgne , T.;
Niessen, J.; Sch eek, S.; Fehlhabe , B.;
Fo s e , M.; S anulla, M.; e al.
GSK3αRegula es Tempo ally
Dynamic Changes in Ribosomal
P o eins upon Amino Acid
S a a ion in Cance Cells. In . J. Mol.
Sci. 2023,24, 13260. h ps://
doi.o g/10.3390/ijms241713260
Academic Edi o : Al ed
King-Yin Lam
Recei ed: 29 June 2023
Re ised: 15 Augus 2023
Accep ed: 18 Augus 2023
Published: 26 Augus 2023
Copy igh : © 2023 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
In e na ional Jou nal o
Molecula Sciences
A icle
GSK3αRegula es Tempo ally Dynamic Changes in Ribosomal
P o eins upon Amino Acid S a a ion in Cance Cells
Lo en Loxha 1,†, Nu ul Khalida Ib ahim 1,† , Anna Sophie S asche 1, Büs a Cina 1, Tim Dolgne 1,
Julia Niessen 1, Sabine Sch eek 1, Bea e Fehlhabe 1, Michael Fo s e 2, Ma in S anulla 1and Lau a Hinze 1,*
1Depa men o Pedia ic Hema ology and Oncology, Hanno e Medical School, 30625 Hanno e , Ge many;
loxha.lo en @mh-hanno e .de (L.L.); ib ahim.nu ul@mh-hanno e .de (N.K.I.);
s asche.anna@mh-hanno e .de (A.S.S.); cina .bues a@mh-hanno e .de (B.C.);
dolgne . im@mh-hanno e .de (T.D.); niessen.julia@mh-hanno e .de (J.N.);
sch eek.sabine@mh-hanno e .de (S.S.); ehlhabe .bea e@mh-hanno e .de (B.F.);
s anulla.ma in@mh-hanno e .de (M.S.)
2Ins i u e o Clinical Molecula Biology, Kiel Uni e si y, 24105 Kiel, Ge many; [email p o ec ed]
*Co espondence: hinze.lau a@mh-hanno e .de
†These au ho s con ibu ed equally o his wo k.
Abs ac :
Amino acid a ailabili y is c ucial o cance cells’ su i abili y. Leukemia and colo ec al
cance cells ha e been shown o esis aspa agine deple ion by u ilizing GSK3-dependen p o ea-
somal deg ada ion, e med he Wn -dependen s abiliza ion o p o eins (Wn /STOP), o eplenish
hei amino acid pool. The inhibi ion o GSK3
α
hal s he sou cing o amino acids, which subse-
quen ly leads o cance cell ulne abili y owa d aspa aginase he apy. Howe e , esis ance owa d
GSK3
α
-media ed p o ein b eakdown can occu , whose unde lying mechanism is poo ly unde -
s ood. He e, we se ou o de ine he mechanisms d i ing dependence owa d his deg ada ion
machine y upon aspa agine s a a ion in cance cells. We show he independence o known s ess
esponse pa hways including he in eg a ed s ess esponse media ed wi h GCN2. Addi ionally,
we demons a e he independence o changes in cell cycle p og ession and exp ession le els o he
aspa agine-syn hesizing enzyme ASNS. Ins ead, RNA sequencing e ealed ha GSK3
α
inhibi ion
and aspa agine s a a ion leads o he empo ally dynamic down egula ion o dis inc ibosomal
p o eins, which ha e been shown o display an i-p oli e a i e unc ions. Using a CRISPR/Cas9
iabili y sc een, we demons a e ha he down egula ion o hese speci ic ibosomal p o eins can
escue cell dea h upon GSK3
α
inhibi ion and aspa agine s a a ion. Thus, ou indings sugges he
i al ole o he p e iously un ecognized egula ion o ibosomal p o eins in b idging GSK3
α
ac i i y
and ole ance o aspa agine s a a ion.
Keywo ds:
GSK3
α
; Wn /STOP; aspa aginase; amino acid s a a ion; me abolism; cance ; acu e
leukemia; colo ec al cance ; ibosomal p o eins; gene egula ion
1. In oduc ion
Cance cells ine i ably encoun e s ess due o excessi e p oli e a ion a es, which aise
he demand o nu ien a ailabili y and p o ein syn hesis. Some cance s, such as acu e
lymphoblas ic leukemia (ALL), depend on aspa agine a ailabili y o main ain cell su i al,
which is exploi ed clinically wi h he use o he bac e ially de i ed enzyme aspa aginase
ha deple es aspa agine [1–4].
Howe e , ole ance o amino acid deple ion can cause cance cell esis ance and hus
ep esen s a majo clinical obs acle. An in-dep h cha ac e iza ion o cellula signaling
pa hways is essen ial o unde s and he egula o y mechanisms o cellula homeos asis in
esponse o amino acid dep i a ion.
In p e ious s udies, we could demons a e ha esis an leukemia cells, as well as
colo ec al cance cells (CRC), ely on GSK3-dependen p o ein deg ada ion as an al e na i e
In . J. Mol. Sci. 2023,24, 13260. h ps://doi.o g/10.3390/ijms241713260 h ps://www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2023,24, 13260 2 o 18
sou ce o amino acids o main ain cellula i ness upon amino acid deple ion. The inhibi ion
o GSK3-dependen p o ein deg ada ion leads o he ac i a ion o a non-canonical b anch
o Wn signaling, e med Wn -dependen s abiliza ion o p o eins (Wn /STOP) [
5
], ha
media es cell dea h in he p esence o amino acid sca ci y [
6
–
8
]. Impo an ly, we ound ha
aspa aginase sensi iza ion is solely dependen on he alpha iso o m o GSK3 [
6
–
8
]. Due o
i s ole in di e en cance en i ies, GSK3
α
hus has a pi o al ole in egula ing he cellula
esponse o amino acid dep i a ion. Howe e , cance cells can de elop ole ance owa d
GSK3
α
inhibi ion and aspa agine deple ion, whose mechanis ic unde pinnings a e no
su icien ly unde s ood. Thus, we se ou o de ine molecula ac o s ha d i e o inhibi
cell dea h upon GSK3αinhibi ion and aspa agine s a a ion in cance cells.
En i onmen al s esso s, such as amino acid sho age, a e well known o cause he
accumula ion o mis olded o un olded p o eins, esul ing in endoplasmic e iculum (ER)
s ess [
9
,
10
]. The un olded p o ein esponse (UPR) is a cellula adap i e esponse ha
e ol ed o es o e p o ein- olding homeos asis by educing p o ein syn hesis and by in-
c easing ER p o ein olding [
11
]. P o ein ubiqui ina ion and p o easomal deg ada ion a e
impo an o he deg ada ion o un olded o damaged p o eins [
12
]. Howe e , despi e i s
link o p o easomal deg ada ion, he ac i a ion o Wn /STOP and aspa aginase ea men
has been shown o no a ec es ablished UPR ma ke s such as XBP1 mRNA splicing and
PERK phospho yla ion [
10
], a guing agains ac i a ion o he UPR esponse as a media o
o cell dea h [7].
One cen al signaling node ha con ols he cellula esponse o amino acid a ailabili y
is he e olu iona ily conse ed kinase GCN2 [
13
,
14
]. The key cha ac e is ic o GCN2 wi hin
he in eg a ed s ess esponse (ISR), a homeos a ic sys em by which euka yo ic cells sense
and espond o s ess-inducing signals, is i s ole as a senso o amino acid deple ion [
14
–
16
].
S ess is hen amelio a ed by a ec ing changes in bo h global p o ein syn hesis and he
exp ession o ce ain key genes o ei he es o e homeos asis o induce apop osis [
11
,
17
].
Depending on he leng h and se e i y o s ess, he esponse can be di ec ly p o-su i al,
ac i a ing genes ha oppose he in inging s ess and p omo e a e u n o homeos asis,
o ins ead can induce apop osis i su i al is no possible [
17
–
20
]. While he ac i a ion o
GCN2 upon s a a ion has been shown o inhibi global p o ein ansla ion, some selec ed
ansc ip s, such as he cellula ansc ip ional ac o ATF4, can display an inc ease in
ansla ion [
21
]. Thus, in o de o espond o amino acid deple ion e ec i ely, amino-acid-
syn hesizing enzymes, such as he aspa agine syn hesizing enzyme ASNS, and anspo e
genes a e unde he con ol o he GCN2-ATF4 pa hway [
22
,
23
]. The GCN2-ATF4 pa hway
is c i ical o umo cell su i al and p oli e a ion when challenged by acu e amino acid
dep i a ion [
18
,
24
]. While he acu e esponse has been ex ensi ely s udied, he ac i a ion
in he p esence o ch onic s ess is less de ined. Upon ch onic s a a ion, he ATF4 axis has
been demons a ed o be p o-apop o ic h ough he up egula ion o CHOP [
25
]. Thus, we
explo ed whe he cell dea h media ed by GSK3
α
inhibi ion and aspa aginase ea men
is dependen on he GCN2 axes. Howe e , we ound no up egula ion o ASNS no
dependence on GCN2 o CHOP ac i i y, indica ing independence om he acu e and
ch onic GCN2-ATF4 b anches.
Ins ead, we show ha GSK3
α
inhibi ion leads o he empo ally dynamic down egula-
ion o ibosomal p o eins upon amino acid s a a ion. Ribosome biogenesis is a highly
coo dina ed p ocess in ol ing he syn hesis and p ocessing o ibosomal RNA ( RNA), he
syn hesis o ibosomal p o eins and hei impo in o he nucleus, he assembly o ibosome
subuni s, and he anspo o he ma u e 40S (composed o RPS) and 60S (composed o
RPL) subuni s in o he cy oplasm [
26
–
28
]. In addi ion o hei s uc u al and egula o y
oles in he ansla ion machine y, RPs can pe o m o he “moonligh ing” ex a- ibosomal
unc ions including he egula ion o cell g ow h, p oli e a ion, and di e en ia ion [
29
].
These unc ions a e de ined based on speci ic in e ac ions be ween RPs wi h non- ibosomal
cellula componen s independen o he ibosome [
30
–
34
]. In he con ex o ex a- ibosomal
unc ions, p e ious s udies could demons a e an in iguing pa e n o RP exp ession in
cance s. While se e al RP genes displayed p o-oncogenic e ec s and esul ed in inc eased
In . J. Mol. Sci. 2023,24, 13260 3 o 18
p oli e a ion, o he RP genes consis en ly exhibi ed nega i e dys egula ion ac oss cance s,
which he eby ac ed di ec ly o indi ec ly as umo supp esso s.
In line, we ound ha inhibi ion o dis inc ibosomal p o eins o he small and la ge
subuni s, which a e known o display an an i-p oli e a i e e ec , could escue GSK3-
inhibi ed cells om aspa aginase-induced cy o oxici y.
Thus, we can demons a e a p e iously un ecognized link be ween ibosomal p o eins
and GSK3αac i i y in egula ing he cellula esponse o amino acid s a a ion.
2. Resul s
2.1. Loss o GSK3αInduces Aspa aginase Cy o oxici y Independen om ASNS Exp ession in
Resis an Cance Cells
To de ine ac o s ha d i e dependence owa d he GSK3
α
-dependen p o easomal
deg ada ion machine y, we s a ed by inducing a knockdown o GSK3
α
in Ju ka T-ALL
cells as well as in he colo ec al cance (CRC) cell line HCT15. The knockdown o GSK3
α
us-
ing wo independen shRNAs in Ju ka (Figu e S1A), as well as in HCT15 cells (
Figu e S1B
),
esul ed in a s iking aspa aginase sensi iza ion (Figu e 1A,B), which could be escued by
exp essing he GSK3
α
wild- ype (WT) sequence (Figu e S1C,D), indica ing an on- a ge
e ec [
7
]. The e ec o he knockdown was also e iden wi h he dec ease in K48-linked
ubiqui in le els (Figu es 1C and S1E), which is one o he well-es ablished hallma ks o
an ac i a ed Wn /STOP pa hway [
5
–
7
,
35
]. In bo h cell lines, he inhibi ion o GSK3
α
and
aspa aginase ea men displayed a obus inc ease in mi ochond ial apop osis, as assessed
wi h Caspase 3/7 ac i i y (Figu e 1D) o BH3 p o iling (Figu es 1E and S1F).
In . J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 o 19
Figu e 1. Loss o GSK3α induces aspa aginase cy o oxici y independen om ASNS exp ession in
esis an cance cells. (A,B) Cells we e ansduced wi h indica ed cons uc s and ea ed wi h ehicle
o 100 U/L o aspa aginase in biological iplica es. Rela i e iabili y was assessed a e 8 days o
ea men by coun ing iable cells. No e ha an ea lie ime poin was chosen as in [6] o allow o
di ec compa ison be ween he wo cell lines o diffe en cance en i ies. S a is ical signi icance was
assessed using a one-way ANOVA wi h Dunne ’s adjus men o mul iple compa isons. (C) Ju ka
cells we e ansduced wi h indica ed shRNAs. Upon knockdown alida ion, p o ein le els o K48-
linked ubiqui in and GAPDH we e assessed using Wes e n blo analysis. (D) Indica ed cells we e
ansduced wi h indica ed shRNAs, ea ed wi h ehicle o 100 U/L o aspa aginase o 48 h, and
Caspase 3/7 ac i i y was assessed in biological iplica es. S a is ical signi icance was assessed using
a wo-sided S uden ’s - es wi h Welch adjus men . (E) Cells we e ansduced wi h indica ed
shRNAs and ea ed wi h 100 U/L o aspa aginase o 48 h, and cy och ome C elease was assessed
in biological iplica es. S a is ical signi icance was assessed using a wo-sided S uden ’s - es wi h
Welch adjus men . (F) Ju ka cells we e ansduced wi h indica ed shRNAs and ea ed wi h ehicle
o 100 U/L o aspa aginase in biological iplica es. Rela i e iabili y was assessed a indica ed ime
poin s by coun ing iable cells. All cell coun s we e no malized o shLuc- ansduced, ehicle- ea ed
cells. (G–J) Cell lines we e ansduced wi h indica ed shRNAs and ea ed wi h ehicle o 100 U/L
o aspa aginase a each indica ed ime poin . Rela i e ASNS exp ession was assessed wi h qRT-PCR
analysis in biological duplica es and no malized o each ehicle condi ion. S a is ical signi icance
was assessed using a wo-sided S uden ’s - es wi h Welch adjus men . **** p ≤ 0.0001, *** p ≤ 0.001,
** p ≤ 0.01, * p < 0.05, and n.s. p ≥ 0.05.
2.2. GSK3α-Media ed Response o Ch onic Amino Acid Dep i a ion Is Independen o he
GCN2-CHOP Axis
Nex , we asked whe he GSK3α inhibi ion media es cance cell dea h in esponse o
aspa agine deple ion h ough di ec p o-apop o ic signaling. P e ious s udies could
demons a e ha uncha ged RNAs, which accumula e in acellula ly du ing amino acid
limi a ion, ac i a e he p o ein kinase GCN2, a well-known egula o o ansla ion in
amino-acid-s a ed cells ha phospho yla es he euka yo ic ini ia ion ac o 2α (eIF2α)
[14,40]. eIF2α phospho yla ion can inhibi global p o ein ansla ion and induce he ans-
la ion o speci ic ansc ip s such as ATF4 [21]. ATF4 can hen unc ion o s imula e he
exp ession o a ge genes [22,23] o inc ease amino acid syn hesis and p o ein olding. In
he con ex o acu e ac i a ion, GCN2 se es as a p o-su i al signal [18,40,41], whils he
effec o a ch onic GCN2 ac i a ion emains ill-de ined.
Howe e , in ou con ex , he ch onic axis is a pe inen aspec o be add essed as he
induc ion o cell dea h upon GSK3α inhibi ion in cance cells in ol es pe sis en
Figu e 1.
Loss o GSK3
α
induces aspa aginase cy o oxici y independen om ASNS exp ession
in esis an cance cells. (
A
,
B
) Cells we e ansduced wi h indica ed cons uc s and ea ed wi h
ehicle o 100 U/L o aspa aginase in biological iplica es. Rela i e iabili y was assessed a e
8 days o ea men by coun ing iable cells. No e ha an ea lie ime poin was chosen as in [
6
] o
allow o di ec compa ison be ween he wo cell lines o di e en cance en i ies. S a is ical signi i-
cance was assessed using a one-way ANOVA wi h Dunne ’s adjus men o mul iple compa isons.
(
C
) Ju ka cells we e ansduced wi h indica ed shRNAs. Upon knockdown alida ion, p o ein le els
o K48-linked ubiqui in and GAPDH we e assessed using Wes e n blo analysis. (
D
) Indica ed cells
we e ansduced wi h indica ed shRNAs, ea ed wi h ehicle o 100 U/L o aspa aginase o 48 h,
In . J. Mol. Sci. 2023,24, 13260 4 o 18
and Caspase 3/7 ac i i y was assessed in biological iplica es. S a is ical signi icance was assessed
using a wo-sided S uden ’s - es wi h Welch adjus men . (E) Cells we e ansduced wi h indica ed
shRNAs and ea ed wi h 100 U/L o aspa aginase o 48 h, and cy och ome C elease was assessed
in biological iplica es. S a is ical signi icance was assessed using a wo-sided S uden ’s - es wi h
Welch adjus men . (
F
) Ju ka cells we e ansduced wi h indica ed shRNAs and ea ed wi h ehicle
o 100 U/L o aspa aginase in biological iplica es. Rela i e iabili y was assessed a indica ed ime
poin s by coun ing iable cells. All cell coun s we e no malized o shLuc- ansduced, ehicle- ea ed
cells. (
G
–
J
) Cell lines we e ansduced wi h indica ed shRNAs and ea ed wi h ehicle o 100 U/L o
aspa aginase a each indica ed ime poin . Rela i e ASNS exp ession was assessed wi h qRT-PCR
analysis in biological duplica es and no malized o each ehicle condi ion. S a is ical signi icance
was assessed using a wo-sided S uden ’s - es wi h Welch adjus men . **** p
≤
0.0001, *** p
≤
0.001,
** p≤0.01, * p< 0.05, and n.s. p≥0.05.
Exp ession le els o he aspa agine-syn hesizing enzyme, ASNS, ha e long been a -
ibu ed o sensi i i y and esis ance o aspa aginase. Howe e , s udies could demons a e
ha ASNS exp ession and aspa aginase esponse a e poo ly co ela ed in human leukemia
cells [
36
–
39
]. In e es ingly, ASNS exp ession has been shown o apidly inc ease h ough
ac i a ion o he ansc ip ion ac o ATF4 as an acu e and immedia e cellula esponse
o amino acid dep i a ion [
22
,
23
]. We hus asked whe he cell dea h in he con ex o
aspa agine sca ci y and loss o GSK3αin ol es changes in he exp ession le els o ASNS.
To add ess his ques ion, we ea ed Ju ka , as well as HCT15 cells, wi h aspa aginase
in he p esence o absence o GSK3
α
inhibi ion and subsequen ly assessed ASNS mRNA
exp ession le els. Gi en he ac ha amino acid dep i a ion has o be p esen o se e al
days o obse e he abo e-desc ibed sensi iza ion pheno ype, we assessed ASNS exp ession
le els no only a an ea ly ime poin bu also a e 56 h, a which we we e able o obse e
a leas 50% cell dea h (Figu e 1F and Figu e S1G). Howe e , we ailed o obse e any
signi ican di e ences in GSK3
α
-inhibi ed cells upon aspa agine deple ion (Figu e 1G–J).
Thus, hese indings collec i ely a gue agains he ole o ASNS exp ession in media ing
GSK3α-dependen aspa aginase cy o oxici y.
2.2. GSK3α-Media ed Response o Ch onic Amino Acid Dep i a ion Is Independen o he
GCN2-CHOP Axis
Nex , we asked whe he GSK3
α
inhibi ion media es cance cell dea h in esponse
o aspa agine deple ion h ough di ec p o-apop o ic signaling. P e ious s udies could
demons a e ha uncha ged RNAs, which accumula e in acellula ly du ing amino acid
limi a ion, ac i a e he p o ein kinase GCN2, a well-known egula o o ansla ion in amino-
acid-s a ed cells ha phospho yla es he euka yo ic ini ia ion ac o 2
α
(eIF2
α
) [
14
,
40
].
eIF2
α
phospho yla ion can inhibi global p o ein ansla ion and induce he ansla ion o
speci ic ansc ip s such as ATF4 [
21
]. ATF4 can hen unc ion o s imula e he exp ession
o a ge genes [
22
,
23
] o inc ease amino acid syn hesis and p o ein olding. In he con ex
o acu e ac i a ion, GCN2 se es as a p o-su i al signal [
18
,
40
,
41
], whils he e ec o a
ch onic GCN2 ac i a ion emains ill-de ined.
Howe e , in ou con ex , he ch onic axis is a pe inen aspec o be add essed as he
induc ion o cell dea h upon GSK3
α
inhibi ion in cance cells in ol es pe sis en aspa agine
deple ion. P olonged s a a ion has been shown o induce apop osis h ough he ac i a ion
o ATF4 (Figu e 2A) [
11
,
20
]. This leads o subsequen up egula ion o he p o-apop o ic
ansc ip ion ac o CHOP wi h a esul ing o ma ion o ATF4-CHOP he e odime s ha
can (i) ac i a e u he downs eam p o-apop o ic a ge s and (ii) d i e p o ein ansla ion
leading o ATP deple ion and cell dea h [
11
,
25
,
42
]. Fo ins ance, glu amine s a a ion in
MYC-media ed neu oblas oma has been shown o induce apop osis h ough he GCN2-
ATF4 b anch [43].
In . J. Mol. Sci. 2023,24, 13260 5 o 18
In . J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 6 o 19
Figu e 2. GSK3α-media ed esponse o ch onic amino acid dep i a ion is independen o he GCN2-
CHOP axis. (A) Schema ic depic ion o he GCN2-CHOP axis in he con ex o ch onic amino acid
dep i a ion. (B) Ju ka cells we e ansduced wi h indica ed shRNAs and ea ed wi h ehicle o
100 U/L o aspa aginase in biological iplica es. Rela i e iabili y was assessed a e 6 days o ea -
men by coun ing iable cells. All cell coun s we e no malized o ehicle- ea ed cells. T ea men
was concluded a an ea lie ime poin due o he oxici y o GCN2 and CHOP knockdown a a la e
ime poin . (C) Ju ka cells we e ansduced wi h indica ed cons uc s and ea ed wi h ehicle o
100 U/L o aspa aginase. Rela i e iabili y was assessed a e 8 days o ea men by coun ing iable
cells. All cell coun s we e no malized o shLuc- ansduced, ehicle- ea ed cells. (D) Ju ka cells
we e ansduced wi h indica ed cons uc s and ea ed wi h indica ed ea men s in biological ip-
lica es. Rela i e iabili y was assessed a e 6 days o ea men by coun ing iable cells. Coun s
we e no malized o ehicle- ea ed cells. (E) HCT15 cells we e ea ed as in (B). (F) HCT15 cells we e
ea ed as in (C). (G) HCT15 cells we e ea ed as in (D). S a is ical signi icance was assessed using
a one-way ANOVA wi h Dunne ’s adjus men o mul iple compa isons. **** p ≤ 0.0001, *** p ≤ 0.001,
** p ≤ 0.01, * p < 0.05, and n.s. p ≥ 0.05.
2.3. Cell Dea h upon Inhibi ion o GSK3α Is No Media ed by Changes in Cell Cycle
The Wn /STOP pa hway is bes known o egula e cell size and g ow h owing o i s
ole in s abilizing p o eins du ing mi osis. A p e ious s udy has shown ha p o eins we e
pe iodically s abilized a he G2/M cell cycle phase when Wn /STOP was ac i e [5]. This
is essen ial o op imal cell cycle p og ession as cells equi e a sufficien amoun o p o-
eins in p epa a ion o cell di ision.
Figu e 2.
GSK3
α
-media ed esponse o ch onic amino acid dep i a ion is independen o he GCN2-
CHOP axis. (
A
) Schema ic depic ion o he GCN2-CHOP axis in he con ex o ch onic amino acid
dep i a ion. (
B
) Ju ka cells we e ansduced wi h indica ed shRNAs and ea ed wi h ehicle o
100 U/L o aspa aginase in biological iplica es. Rela i e iabili y was assessed a e 6 days o
ea men by coun ing iable cells. All cell coun s we e no malized o ehicle- ea ed cells. T ea men
was concluded a an ea lie ime poin due o he oxici y o GCN2 and CHOP knockdown a a
la e ime poin . (
C
) Ju ka cells we e ansduced wi h indica ed cons uc s and ea ed wi h ehicle
o 100 U/L o aspa aginase. Rela i e iabili y was assessed a e 8 days o ea men by coun ing
iable cells. All cell coun s we e no malized o shLuc- ansduced, ehicle- ea ed cells. (
D
) Ju ka
cells we e ansduced wi h indica ed cons uc s and ea ed wi h indica ed ea men s in biological
iplica es. Rela i e iabili y was assessed a e 6 days o ea men by coun ing iable cells. Coun s
we e no malized o ehicle- ea ed cells. (
E
) HCT15 cells we e ea ed as in (
B
). (
F
) HCT15 cells we e
ea ed as in (
C
). (
G
) HCT15 cells we e ea ed as in (
D
). S a is ical signi icance was assessed using a
one-way ANOVA wi h Dunne ’s adjus men o mul iple compa isons. **** p
≤
0.0001, *** p
≤
0.001,
** p≤0.01, * p< 0.05, and n.s. p≥0.05.
Thus, we i s wonde ed whe he cell dea h in esponse o he loss o GSK3
α
and
amino acid sca ci y is media ed h ough a GCN2-CHOP-dependen axis. To es his ex-
pe imen ally, we s a ed by inducing a knockdown o wo downs eam e ec o s o he
axis, GCN2 and CHOP, in Ju ka T-ALL cells (Figu e S1H). CHOP se es as he majo
p o-apop o ic e ec o o ATF4 ac i a ion in he ER s ess esponse [
44
,
45
]. We hus ea-
soned ha i cell dea h is media ed h ough his axis, a knockdown o he key e ec o s
should block aspa aginase sensi iza ion induced by he inhibi ion o GSK3
α
. Howe e , he

In . J. Mol. Sci. 2023,24, 13260 6 o 18
knockdown o CHOP o GCN2 ailed o escue shGSK3
α
cells om aspa aginase-induced
cell dea h (Figu e 2B). By con as , exp ession o he hype ac i e p o easomal subuni
∆
N-PSMA4, which di ec ly s imula es p o easomal deg ada ion o a ange o p o easomal
subs a es [
46
], se ed as a posi i e con ol [
6
,
7
] and was able o escue shGSK3
α
cells
om aspa aginase cy o oxici y (Figu e 2C). Impo an ly, all desc ibed indings could be
independen ly alida ed in he colo ec al cance cell line HCT15 (Figu es 2E,F and S1I).
Second, o u he s eng hen he a gumen ha GSK3
α
-media ed aspa aginase e-
sponse is independen o he ATF4-CHOP b anch, we asked whe he inhibi ion o p o ein
syn hesis can p o ec cells om he oxici y o GSK3
α
inhibi ion and aspa agine deple ion.
This is due o he ac ha ATF4-CHOP he e odime s can d i e p o ein ansla ion leading
o ATP deple ion and cell dea h [
25
]. Howe e , ea men wi h he elonga ion inhibi o
homoha ing onine ailed o escue cells om GSK3
α
-media ed cell dea h upon aspa agine
deple ion in Ju ka T-ALL cells (Figu e 2D) as well as in colo ec al cance cells (Figu e 2G).
Collec i ely, hese da a indica e ha GSK3
α
inhibi ion media es aspa aginase sensi iza ion
independen o he GCN2-CHOP axis.
2.3. Cell Dea h upon Inhibi ion o GSK3αIs No Media ed by Changes in Cell Cycle
The Wn /STOP pa hway is bes known o egula e cell size and g ow h owing o i s
ole in s abilizing p o eins du ing mi osis. A p e ious s udy has shown ha p o eins we e
pe iodically s abilized a he G2/M cell cycle phase when Wn /STOP was ac i e [
5
]. This
is essen ial o op imal cell cycle p og ession as cells equi e a su icien amoun o p o eins
in p epa a ion o cell di ision.
Thus, we asked whe he dis inc changes in he cell cycle could p omp he p og essi e
di ec ion o cell dea h. To es his, we de e mined cell cycle s ages in Ju ka and HCT15
cells ansduced wi h GSK3
α
shRNA. Howe e , we did no ind any signi ican e ec s in
ei he he p esence o absence o aspa agine s a a ion (Figu e 3A–D). This indica es ha
cell cycle changes do no in luence he cou se owa d cell dea h.
In . J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 7 o 19
Thus, we asked whe he dis inc changes in he cell cycle could p omp he p og es-
si e di ec ion o cell dea h. To es his, we de e mined cell cycle s ages in Ju ka and
HCT15 cells ansduced wi h GSK3α shRNA. Howe e , we did no ind any signi ican
effec s in ei he he p esence o absence o aspa agine s a a ion (Figu e 3A–D). This in-
dica es ha cell cycle changes do no in luence he cou se owa d cell dea h.
Figu e 3. GSK3α inhibi ion and aspa agine deple ion do no cause changes in he cell cycle. (A)
Ju ka cells we e ansduced wi h indica ed shRNAs and ea ed wi h ehicle o 100 U/L o aspa a-
ginase. Cell cycle analysis was conduc ed a e 48 h o ea men using low cy ome y in biological
duplica es. (B) S a is ical analysis o he cell cycle analysis om (A) o each cell cycle phase. S a is-
ical signi icance was assessed using a one-way ANOVA wi h Dunne ’s adjus men o mul iple
compa isons. (C) HCT15 cells we e ea ed and assessed as in (A). (D) S a is ical analysis o he cell
cycle analysis om (C) o each cell cycle phase assessed as in (B). n.s. p ≥ 0.05.
2.4. Inhibi ion o GSK3α Leads o Tempo ally Dynamic Down egula ion o Dis inc Ribosomal
P o eins in he P esence o Aspa agine Dep i a ion
Nex , we aimed o iden i y genes and biological p ocesses egula ed upon inhibi ion
o GSK3α. In an explo a o y app oach, we i s induced a obus GSK3α knockdown in
Ju ka T-ALL cells and subsequen ly ea ed hese cells wi h ehicle o aspa aginase. To
iden i y ea ly, in e media e, and la e esponses o GSK3α inhibi ion in he p esence o
absence o aspa agine deple ion, we ha es ed cells a 8, 16, 32, and 56 h o ea men
(Figu e 4A) and pe o med gene exp ession analysis wi h RNA-sequencing. We chose
hese ime poin s due o he g adual dec ease in cell iabili y (Figu es 1F and S1G). This
allowed he de ec ion o ea ly changes due o induc ion o cell dea h as well as changes a
la e ime poin s wi h a small subse o emaining su i ing cells.
Analysis o he RNA-sequencing esul s e ealed ha he exp ession o 2046 an-
sc ip s changed as ea ly as 8 h a e he s a o aspa aginase ea men . A e 56 h o ea -
men , 1161 and 880 ansc ip s we e diffe en ially up egula ed o down egula ed, espec-
i ely ( old change > 1.5). In e es ingly, he absolu e numbe o diffe en ially exp essed
ansc ip s did no g ow signi ican ly o e ime, while he cons ella ion o ansc ip s ha
we e diffe en ially exp essed changed be ween ime poin s (Figu e S2A).
Figu e 3.
GSK3
α
inhibi ion and aspa agine deple ion do no cause changes in he cell cycle. (
A
) Ju ka
cells we e ansduced wi h indica ed shRNAs and ea ed wi h ehicle o 100 U/L o aspa aginase.
Cell cycle analysis was conduc ed a e 48 h o ea men using low cy ome y in biological duplica es.
In . J. Mol. Sci. 2023,24, 13260 7 o 18
(
B
) S a is ical analysis o he cell cycle analysis om (
A
) o each cell cycle phase. S a is ical signi i-
cance was assessed using a one-way ANOVA wi h Dunne ’s adjus men o mul iple compa isons.
(
C
) HCT15 cells we e ea ed and assessed as in (
A
). (
D
) S a is ical analysis o he cell cycle analysis
om (C) o each cell cycle phase assessed as in (B). n.s. p≥0.05.
2.4. Inhibi ion o GSK3αLeads o Tempo ally Dynamic Down egula ion o Dis inc Ribosomal
P o eins in he P esence o Aspa agine Dep i a ion
Nex , we aimed o iden i y genes and biological p ocesses egula ed upon inhibi ion o
GSK3
α
. In an explo a o y app oach, we i s induced a obus GSK3
α
knockdown in Ju ka
T-ALL cells and subsequen ly ea ed hese cells wi h ehicle o aspa aginase. To iden i y
ea ly, in e media e, and la e esponses o GSK3
α
inhibi ion in he p esence o absence o
aspa agine deple ion, we ha es ed cells a 8, 16, 32, and 56 h o ea men (Figu e 4A) and
pe o med gene exp ession analysis wi h RNA-sequencing. We chose hese ime poin s due
o he g adual dec ease in cell iabili y (Figu es 1F and S1G). This allowed he de ec ion o
ea ly changes due o induc ion o cell dea h as well as changes a la e ime poin s wi h a
small subse o emaining su i ing cells.
In . J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 9 o 19
Figu e 4. Inhibi ion o GSK3α leads o empo ally dynamic down egula ion o dis inc ibosomal
p o eins in he p esence o aspa agine dep i a ion. (A) Schema ic depic ion o he wo k low o ob-
ain samples o RNA sequencing in Ju ka T-ALL cells. Cells ansduced wi h indica ed shRNAs
we e ea ed wi h ehicle o 100 U/L o aspa aginase and sampled a each ime poin indica ed. (B)
Sca e plo showing he log2FC o compa isons be ween he un ea ed 0 h ime poin and ea men
o all ime poin s. A log2FC cu -off o 1.5 was used o iden i y diffe en ially exp essed ansc ip s,
indica ed wi h dashed lines. Red do s show RP ansc ip s ha a e diffe en ially down egula ed in
shGSK3α cells while no being down egula ed in shLuc cells. Blue do s indica e RP ansc ip s ha
we e s udied u he . (C) Rspo3; K as; and T p53 mouse in es inal o ganoids we e ea ed con inu-
ously wi h ehicle o aspa aginase o 14 days. Upon ou g ow h o o ganoids in he aspa aginase-
ea ed condi ions, o ganoids we e ha es ed and analyzed wi h RNA sequencing. (D) Sca e plo
showing diffe en ially down egula ed ansc ip s in o ganoids om (C). Red do s indica e RP an-
sc ip s. Blue do s indica e RP ansc ip s ha we e independen ly alida ed in he CRISPR/Cas9
sc een (Figu e 5).
2.5. Inhibi ion o Speci ic Ribosomal P o eins P omo es Cellula Fi ness upon GSK3α Inhibi ion
and Aspa agine S a a ion
To in es iga e he ole o RPS/RPL in media ing cellula i ness in he con ex o
GSK3α inhibi ion and aspa aginase ea men , we gene a ed GSK3α knockou (KO) as
well as AAVS1 sa e ha bo con ol single-cell clones in Ju ka T-ALL cells (Figu e S2B,C).
O no e, we picked GSK3α KO single-cell clones wi h an in e media e aspa aginase sensi-
iza ion o allow sc eening o bo h sgRNA en ichmen ( esis ance) and sgRNA d opou
(exace ba ed sensi iza ion). Upon iden i ica ion o sui able single-cell clones, we ans-
duced hese cells wi h a genome-wide sgRNA lib a y (B unello loss o unc ion lib a y),
ollowed by ea men wi h ehicle o aspa aginase (Figu e S2D). Analysis o sgRNA ep-
esen a ion e ealed a signi ican en ichmen o sgRNAs a ge ing ibosomal p o eins
when compa ing GSK3α KO cells o AAVS1 cells in he p esence o aspa aginase ea -
men (p = 3.64 × 10−7, Fishe ’s exac es ) (Figu e 5A,B, Tables S3 and S4).
Figu e 4.
Inhibi ion o GSK3
α
leads o empo ally dynamic down egula ion o dis inc ibosomal
p o eins in he p esence o aspa agine dep i a ion. (
A
) Schema ic depic ion o he wo k low o
ob ain samples o RNA sequencing in Ju ka T-ALL cells. Cells ansduced wi h indica ed shRNAs
we e ea ed wi h ehicle o 100 U/L o aspa aginase and sampled a each ime poin indica ed.
(
B
) Sca e plo showing he log2FC o compa isons be ween he un ea ed 0 h ime poin and ea men
o all ime poin s. A log2FC cu -o o 1.5 was used o iden i y di e en ially exp essed ansc ip s,
indica ed wi h dashed lines. Red do s show RP ansc ip s ha a e di e en ially down egula ed in
shGSK3αcells while no being down egula ed in shLuc cells. Blue do s indica e RP ansc ip s ha
In . J. Mol. Sci. 2023,24, 13260 8 o 18
we e s udied u he . (
C
) Rspo3; K as; and T p53 mouse in es inal o ganoids we e ea ed con inu-
ously wi h ehicle o aspa aginase o 14 days. Upon ou g ow h o o ganoids in he aspa aginase-
ea ed condi ions, o ganoids we e ha es ed and analyzed wi h RNA sequencing. (
D
) Sca e plo
showing di e en ially down egula ed ansc ip s in o ganoids om (
C
). Red do s indica e RP an-
sc ip s. Blue do s indica e RP ansc ip s ha we e independen ly alida ed in he CRISPR/Cas9
sc een (Figu e 5).
In . J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 10 o 19
Fo alida ion o selec i e RPS and RPL om bo h he ansc ip omic app oaches and
he CRISPR/Cas9 sc een, we ocused on he op hi s RPS27, RPL6, and RPL36, which ha e
been shown o display an i-p oli e a i e pheno ypes, pa ially h ough a p53-dependen
mechanism [47,49,50].
To alida e ha loss o RPL/RPS con e s a su i al ad an age in GSK3α-inhibi ed
cells, we len i i ally ansduced sgRNAs a ge ing RPS27 and RPL6 in AAVS1 as well as
wo independen GSK3α KO single-cell clones (Figu e S2B–D). Efficien gene silencing
was con i med wi h a qRT-PCR (Figu e S2E). Indeed, he inhibi ion o RPL/RPS wi h sgR-
NAs was able o block GSK3α-inhibi ion media ed aspa aginase sensi iza ion (Figu e 5C).
In line, he knockdown o RPL36 in T-ALL as well as in CRC cells could block aspa agi-
nase cy o oxici y upon GSK3α inhibi ion and aspa agine s a a ion (Figu es 5D,E and
S2F).
These esul s unde line ha loss o dis inc RPS/RPL can con e a su i al ad an age
in he con ex o GSK3α inhibi ion and aspa agine s a a ion. O no e, ou desc ibed ind-
ings a e in line wi h p e iously published da a showing ha posi i ely selec ed sgRNAs
a ge p e e en ially RPS/RPL ha a e known o be down egula ed in cance cells due o
hei an i-p oli e a i e effec [47,48].
Taken oge he , ou indings sugges he i al ole o he p e iously un ecognized
egula ion o ibosomal p o eins in b idging GSK3α ac i i y and ole ance o aspa agine
s a a ion.
Figu e 5. Inhibi ion o speci ic ibosomal p o eins p omo es cellula i ness upon GSK3α inhibi ion
and aspa agine s a a ion. (A) Schema ic wo k low o he CRISPR/Cas9 sc een. (B) Top 15 genes
ha a e diffe en ially affec ed in amino-acid-dep i ed condi ions be ween he wo indica ed cell
lines om he expe imen shown in (A). Ribosomal p o eins a e highligh ed in blue. (C) GSK3α-KO
single-cell clones we e ansduced wi h indica ed sgRNAs and ea ed wi h ehicle o 100 U/L o
aspa aginase in biological duplica es. Rela i e iabili y was assessed a e 6 days o ea men by
coun ing iable cells. All cell coun s we e no malized o ehicle- ea ed cells. No e ha aspa aginase
sensi iza ion was no as s iking because clones we e chosen based on an in e media e esponse o
he genome-wide CRISPR/Cas9 sc een. S a is ical signi icance was assessed using a one-way
ANOVA wi h Dunne ’s adjus men o mul iple compa isons. (D,E) Cells we e ansduced wi h
indica ed shRNAs and ea ed wi h ehicle o 100 U/L o aspa aginase in biological duplica es.
Figu e 5.
Inhibi ion o speci ic ibosomal p o eins p omo es cellula i ness upon GSK3
α
inhibi ion
and aspa agine s a a ion. (
A
) Schema ic wo k low o he CRISPR/Cas9 sc een. (
B
) Top 15 genes
ha a e di e en ially a ec ed in amino-acid-dep i ed condi ions be ween he wo indica ed cell
lines om he expe imen shown in (
A
). Ribosomal p o eins a e highligh ed in blue. (
C
) GSK3
α
-KO
single-cell clones we e ansduced wi h indica ed sgRNAs and ea ed wi h ehicle o 100 U/L o
aspa aginase in biological duplica es. Rela i e iabili y was assessed a e 6 days o ea men by
coun ing iable cells. All cell coun s we e no malized o ehicle- ea ed cells. No e ha aspa aginase
sensi iza ion was no as s iking because clones we e chosen based on an in e media e esponse
o he genome-wide CRISPR/Cas9 sc een. S a is ical signi icance was assessed using a one-way
ANOVA wi h Dunne ’s adjus men o mul iple compa isons. (
D
,
E
) Cells we e ansduced wi h
indica ed shRNAs and ea ed wi h ehicle o 100 U/L o aspa aginase in biological duplica es.
Rela i e iabili y was assessed a e 6 days o ea men by coun ing iable cells. All cell coun s
we e no malized o ehicle- ea ed cells. No e ha aspa aginase sensi iza ion was no as s iking
because clones we e chosen based on an in e media e esponse o he genome-wide CRISPR/Cas9
sc een. S a is ical signi icance was assessed using a one-way ANOVA wi h Dunne ’s adjus men o
mul iple compa isons. **** p≤0.0001, *** p≤0.001, ** p≤0.01, * p< 0.05, and n.s. p≥0.05.
Analysis o he RNA-sequencing esul s e ealed ha he exp ession o 2046 ansc ip s
changed as ea ly as 8 h a e he s a o aspa aginase ea men . A e 56 h o ea men ,
1161 and 880 ansc ip s we e di e en ially up egula ed o down egula ed, espec i ely
( old change > 1.5). In e es ingly, he absolu e numbe o di e en ially exp essed ansc ip s
did no g ow signi ican ly o e ime, while he cons ella ion o ansc ip s ha we e
di e en ially exp essed changed be ween ime poin s (Figu e S2A).
In . J. Mol. Sci. 2023,24, 13260 9 o 18
Upon u he analysis o di e en ially exp essed ansc ip s, we obse ed ha ibo-
somal p o eins o he small (RPS) and la ge (RPL) subuni s we e signi ican ly en iched
( ishe p7.44
×
10
−10
a 56 h aspa aginase ea men ) in down egula ed ansc ip s in
he p esence o an aspa agine deple ion in shGSK3
α
cells when compa ed o shLuc cells
( old change < −1.5 in shGSK3αwhile old change > −1.5 in shLuc) (Figu e 4B, Table S1).
In iguingly, besides hei ole in he assembly o ibosomal componen s, ibosomal
p o eins can also pe o m o he ex a- ibosomal unc ions, including he egula ion o cell
p oli e a ion [
29
–
34
]. These unc ions a e de ined based on speci ic in e ac ions be ween
RPs wi h non- ibosomal cellula componen s independen o he ibosome [
30
–
34
]. As
desc ibed abo e, we obse ed a GCN2-CHOP independen pheno ype and no e ec wi h
he elonga ion inhibi o homoha ing onine, indica ing ha d i ing p o ein ansla ion wi h
subsequen ATP deple ion is unlikely o cause cell dea h in GSK3
α
-inhibi ed cells. Thus, he
ex a- ibosomal unc ions o RPs appea ed o be an in e es ing axis o
u he in es iga ion.
In he con ex o ex a- ibosomal unc ions, p e ious s udies could demons a e an
in iguing pa e n o RP exp ession in cance s. While some RP genes display p o-oncogenic
e ec s, o he RP genes can ac di ec ly o indi ec ly as umo supp esso s [
47
]. Fo ins ance,
some RP gene knockou s ha e been posi i ely selec ed in a CRISPR-based iabili y sc een
ca ied ou in a melanoma cance cell line [
48
], indica ing ha RP gene loss is no always
de imen al o cellula i ness. Thus, he loss o indi idual ibosomal p o eins co ela es
wi h, and in some cases induces, speci ic e ec s on cellula p oli e a ion.
Thus, we wonde ed whe he he down egula ion o RPS/RPL ansc ip s e lec s a
mechanism in cells ha can su i e amino-acid-dep i ed condi ions in GSK3
α
-inhibi ed cells.
To add ess his ques ion, we u ned o he P p k–Rspo3 usion o CRC o ganoids,
which po en ia e Wn ligand-induced inhibi ion o GSK3. These cells a e known o be
highly aspa aginase sensi i e a baseline bu can de elop esis ance upon con inuous
and p olonged ea men p essu e wi h aspa aginase. Le e aging ou g own P p k–Rspo3
o ganoids upon aspa aginase ea men o RNA sequencing (Figu e 4C), we could eca-
pi ula e ou indings wi h a end owa ds en ichmen o RPL/RPS in down egula ed
ansc ip s (p= 0.1, Fishe ’s exac es ) when compa ed o ehicle- ea ed o ganoids
(Figu e 4D, Table S2).
2.5. Inhibi ion o Speci ic Ribosomal P o eins P omo es Cellula Fi ness upon GSK3αInhibi ion
and Aspa agine S a a ion
To in es iga e he ole o RPS/RPL in media ing cellula i ness in he con ex o
GSK3
α
inhibi ion and aspa aginase ea men , we gene a ed GSK3
α
knockou (KO) as well
as AAVS1 sa e ha bo con ol single-cell clones in Ju ka T-ALL cells (Figu e S2B,C). O no e,
we picked GSK3
α
KO single-cell clones wi h an in e media e aspa aginase sensi iza ion o
allow sc eening o bo h sgRNA en ichmen ( esis ance) and sgRNA d opou (exace ba ed
sensi iza ion). Upon iden i ica ion o sui able single-cell clones, we ansduced hese
cells wi h a genome-wide sgRNA lib a y (B unello loss o unc ion lib a y), ollowed by
ea men wi h ehicle o aspa aginase (Figu e S2D). Analysis o sgRNA ep esen a ion
e ealed a signi ican en ichmen o sgRNAs a ge ing ibosomal p o eins when compa ing
GSK3
α
KO cells o AAVS1 cells in he p esence o aspa aginase ea men (p= 3.64
×
10
−7
,
Fishe ’s exac es ) (Figu e 5A,B, Tables S3 and S4).
Fo alida ion o selec i e RPS and RPL om bo h he ansc ip omic app oaches and
he CRISPR/Cas9 sc een, we ocused on he op hi s RPS27, RPL6, and RPL36, which ha e
been shown o display an i-p oli e a i e pheno ypes, pa ially h ough a p53-dependen
mechanism [47,49,50].
To alida e ha loss o RPL/RPS con e s a su i al ad an age in GSK3
α
-inhibi ed
cells, we len i i ally ansduced sgRNAs a ge ing RPS27 and RPL6 in AAVS1 as well as
wo independen GSK3
α
KO single-cell clones (Figu e S2B–D). E icien gene silencing was
con i med wi h a qRT-PCR (Figu e S2E). Indeed, he inhibi ion o RPL/RPS wi h sgRNAs
was able o block GSK3
α
-inhibi ion media ed aspa aginase sensi iza ion (Figu e 5C). In
In . J. Mol. Sci. 2023,24, 13260 16 o 18
Acknowledgmen s:
We hank Lukas E. Dow o he o ganoid models and Min Jae Lee o p o iding
he hype ac i e p o easomal subuni .
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Re e ences
1.
DeAngelo, D.J.; S e enson, K.E.; Dahlbe g, S.E.; Sil e man, L.B.; Couban, S.; Supko, J.G.; Am ein, P.C.; Ballen, K.K.; Se el, M.D.;
Tu ne , A.R.; e al. Long- e m ou come o a pedia ic-inspi ed egimen used o adul s aged 18-50 yea s wi h newly diagnosed
acu e lymphoblas ic leukemia. Leukemia 2015,29, 526–534. [C ossRe ]
2.
Place, A.E.; S e enson, K.E.; V ooman, L.M.; Ha is, M.H.; Hun , S.K.; O’B ien, J.E.; Supko, J.G.; Asselin, B.L.; A hale, U.H.;
Cla ell, L.A.; e al. In a enous pegyla ed aspa aginase e sus in amuscula na i e Esche ichia coli L-aspa aginase in newly
diagnosed childhood acu e lymphoblas ic leukaemia (DFCI 05-001): A andomised, open-label phase 3 ial. Lance Oncol.
2015
,
16, 1677–1690. [C ossRe ] [PubMed]
3.
We zle , M.; San o d, B.L.; Ku zbe g, J.; DeOli ei a, D.; F ankel, S.R.; Powell, B.L.; Koli z, J.E.; Bloom ield, C.D.; La son, R.A.
E ec i e aspa agine deple ion wi h pegyla ed aspa aginase esul s in imp o ed ou comes in adul acu e lymphoblas ic leukemia:
Cance and Leukemia G oup B S udy 9511. Blood 2007,109, 4164–4167. [C ossRe ] [PubMed]
4.
Pession, A.; Valsecchi, M.G.; Mase a, G.; Kamps, W.A.; Magya osy, E.; Rizza i, C.; an We ing, E.R.; Lo Nig o, L.; an de Does,
A.; Loca elli, F.; e al. Long- e m esul s o a andomized ial on ex ended use o high dose L-aspa aginase o s anda d isk
childhood acu e lymphoblas ic leukemia. J. Clin. Oncol. 2005,23, 7161–7167. [C ossRe ]
5.
Aceb on, S.P.; Ka aulano , E.; Be ge , B.S.; Huang, Y.L.; Nieh s, C. Mi o ic wn signaling p omo es p o ein s abiliza ion and
egula es cell size. Mol. Cell 2014,54, 663–674. [C ossRe ] [PubMed]
6.
Hinze, L.; Lab osse, R.; Dega , J.; Han, T.; Scha o , E.M.; Sch eek, S.; Ka im, S.; McGuckin, C.; Sache , J.R.; Wagne , F.; e al.
Exploi ing he The apeu ic In e ac ion o WNT Pa hway Ac i a ion and Aspa aginase o Colo ec al Cance The apy. Cance
Disco . 2020,10, 1690–1705. [C ossRe ] [PubMed]
7.
Hinze, L.; P i mann, M.; Ka im, S.; Dega , J.; McGuckin, C.; Vinjamu , D.; Sache , J.; S e enson, K.E.; Neube g, D.S.;
O ellana, E.; e al. Syn he ic Le hali y o Wn Pa hway Ac i a ion and Aspa aginase in D ug-Resis an Acu e Leukemias.
Cance Cell 2019,35, 664–676.e7. [C ossRe ] [PubMed]
8.
Hinze, L.; Sch eek, S.; Zeug, A.; Ib ahim, N.K.; Fehlhabe , B.; Loxha, L.; Cina , B.; Ponimaskin, E.; Dega , J.; McGuckin, C.; e al.
Sup amolecula assembly o GSK3
α
as a cellula esponse o amino acid s a a ion. Mol. Cell
2022
,82, 2858–2870.e8. [C ossRe ]
9.
Wang, M.; Kau man, R.J. The impac o he endoplasmic e iculum p o ein- olding en i onmen on cance de elopmen . Na . Re .
Cance 2014,14, 581–597. [C ossRe ] [PubMed]
10.
Wal e , P.; Ron, D. The un olded p o ein esponse: F om s ess pa hway o homeos a ic egula ion. Science
2011
,334, 1081–1086.
[C ossRe ]
11.
Wang, M.; Kau man, R.J. P o ein mis olding in he endoplasmic e iculum as a condui o human disease. Na u e
2016
,529,
326–335. [C ossRe ]
12.
Kwon, Y.T.; Ciechano e , A. The Ubiqui in Code in he Ubiqui in-P o easome Sys em and Au ophagy. T ends Biochem. Sci.
2017
,
42, 873–886. [C ossRe ] [PubMed]
13.
B öe , S.; B öe , A. Amino acid homeos asis and signalling in mammalian cells and o ganisms. Biochem. J.
2017
,474, 1935–1963.
[C ossRe ]
14.
Wek, S.A.; Zhu, S.; Wek, R.C. The his idyl- RNA syn he ase- ela ed sequence in he eIF-2 alpha p o ein kinase GCN2 in e ac s
wi h RNA and is equi ed o ac i a ion in esponse o s a a ion o di e en amino acids. Mol. Cell. Biol.
1995
,15, 4497–4506.
[C ossRe ] [PubMed]
15.
Gold, L.T.; Masson, G.R. GCN2: Roles in umou de elopmen and p og ession. Biochem. Soc. T ans.
2022
,50, 737–745. [C ossRe ]
16.
Donnelly, N.; Go man, A.M.; Gup a, S.; Samali, A. The eIF2
α
kinases: Thei s uc u es and unc ions. Cell. Mol. Li e Sci.
2013
,70,
3493–3511. [C ossRe ]
17.
Pakos-Zeb ucka, K.; Ko yga, I.; Mnich, K.; Ljujic, M.; Samali, A.; Go man, A.M. The in eg a ed s ess esponse. EMBO Rep.
2016
,
17, 1374–1395. [C ossRe ]
18.
Ye, J.; Kumano a, M.; Ha , L.S.; Sloane, K.; Zhang, H.; De Panis, D.N.; Bob o niko a-Ma jon, E.; Diehl, J.A.; Ron, D.; Koumenis,
C. The GCN2-ATF4 pa hway is c i ical o umou cell su i al and p oli e a ion in esponse o nu ien dep i a ion. EMBO J.
2010,29, 2082–2096. [C ossRe ] [PubMed]
19.
Nikono o a, I.A.; Mi ek, E.T.; Signo e, C.C.; Goudie, M.P.; Wek, R.C.; An hony, T.G. Time- esol ed analysis o amino acid s ess
iden i ies eIF2 phospho yla ion as necessa y o inhibi mTORC1 ac i i y in li e . J. Biol. Chem. 2018,293, 5005–5015. [C ossRe ]
20.
Ru kowski, D.T.; A nold, S.M.; Mille , C.N.; Wu, J.; Li, J.; Gunnison, K.M.; Mo i, K.; Sadighi Akha, A.A.; Raden, D.; Kau man, R.J.
Adap a ion o ER s ess is media ed by di e en ial s abili ies o p o-su i al and p o-apop o ic mRNAs and p o eins. PLoS Biol.
2006,4, e374. [C ossRe ] [PubMed]
21.
Ha ding, H.P.; No oa, I.; Zhang, Y.; Zeng, H.; Wek, R.; Schapi a, M.; Ron, D. Regula ed ansla ion ini ia ion con ols s ess-
induced gene exp ession in mammalian cells. Mol. Cell 2000,6, 1099–1108. [C ossRe ]
22.
Siu, F.; Bain, P.J.; LeBlanc-Cha in, R.; Chen, H.; Kilbe g, M.S. ATF4 is a media o o he nu ien -sensing esponse pa hway ha
ac i a es he human aspa agine syn he ase gene. J. Biol. Chem. 2002,277, 24120–24127. [C ossRe ]

In . J. Mol. Sci. 2023,24, 13260 17 o 18
23.
Chen, H.; Pan, Y.X.; Dudenhausen, E.E.; Kilbe g, M.S. Amino acid dep i a ion induces he ansc ip ion a e o he human
aspa agine syn he ase gene h ough a imed p og am o exp ession and p omo e binding o nu ien - esponsi e basic e-
gion/leucine zippe ansc ip ion ac o s as well as localized his one ace yla ion. J. Biol. Chem.
2004
,279, 50829–50839. [PubMed]
24.
Wang, Y.; Ning, Y.; Alam, G.N.; Jankowski, B.M.; Dong, Z.; Nö , J.E.; Pol e ini, P.J. Amino acid dep i a ion p omo es umo
angiogenesis h ough he GCN2/ATF4 pa hway. Neoplasia 2013,15, 989–997. [C ossRe ]
25.
Han, J.; Back, S.H.; Hu , J.; Lin, Y.H.; Gilde slee e, R.; Shan, J.; Yuan, C.L.; K okowski, D.; Wang, S.; Ha zoglou, M.; e al.
ER-s ess-induced ansc ip ional egula ion inc eases p o ein syn hesis leading o cell dea h. Na . Cell Biol.
2013
,15, 481–490.
[C ossRe ] [PubMed]
26.
an Riggelen, J.; Ye il, A.; Felshe , D.W. MYC as a egula o o ibosome biogenesis and p o ein syn hesis. Na . Re . Cance
2010
,
10, 301–309. [C ossRe ]
27.
Linds öm, M.S. Eme ging unc ions o ibosomal p o eins in gene-speci ic ansc ip ion and ansla ion. Biochem. Biophys. Res.
Commun. 2009,379, 167–170. [C ossRe ]
28.
Rodnina, M.V.; Win e meye , W. Recen mechanis ic insigh s in o euka yo ic ibosomes. Cu . Opin. Cell Biol.
2009
,21, 435–443.
[C ossRe ]
29.
Weisbe g, R.A. T ansc ip ion by moonligh : S uc u al basis o an ex a ibosomal ac i i y o ibosomal p o ein S10. Mol. Cell
2008
,
32, 747–748. [C ossRe ]
30. Wool, I.G. Ex a ibosomal unc ions o ibosomal p o eins. T ends Biochem. Sci. 1996,21, 164–165. [C ossRe ] [PubMed]
31.
Wang, W.; Nag, S.; Zhang, X.; Wang, M.H.; Wang, H.; Zhou, J.; Zhang, R. Ribosomal p o eins and human diseases: Pa hogenesis,
molecula mechanisms, and he apeu ic implica ions. Med. Res. Re . 2015,35, 225–285. [C ossRe ]
32.
Wa ne , J.R.; McIn osh, K.B. How common a e ex a ibosomal unc ions o ibosomal p o eins? Mol. Cell
2009
,34, 3–11. [C ossRe ]
33.
Kang, J.; B ajano ski, N.; Chan, K.T.; Xuan, J.; Pea son, R.B.; Sanij, E. Ribosomal p o eins and human diseases: Molecula
mechanisms and a ge ed he apy. Signal T ansduc . Ta ge . The . 2021,6, 323. [C ossRe ]
34.
Komili, S.; Fa ny, N.G.; Ro h, F.P.; Sil e , P.A. Func ional speci ici y among ibosomal p o eins egula es gene exp ession. Cell
2007,131, 557–571. [C ossRe ] [PubMed]
35.
Taelman, V.F.; Dob owolski, R.; Plouhinec, J.L.; Fuen ealba, L.C.; Vo wald, P.P.; Gumpe , I.; Saba ini, D.D.; De Robe is, E.M.
Wn signaling equi es seques a ion o glycogen syn hase kinase 3 inside mul i esicula endosomes. Cell
2010
,143, 1136–1148.
[C ossRe ] [PubMed]
36.
He mano a, I.; Zalio a, M.; T ka, J.; S a ko a, J. Low exp ession o aspa agine syn he ase in lymphoid blas s p ecludes i s ole in
sensi i i y o L-aspa aginase. Exp. Hema ol. 2012,40, 657–665. [C ossRe ] [PubMed]
37.
Holleman, A.; Cheok, M.H.; den Boe , M.L.; Yang, W.; Vee man, A.J.; Kazemie , K.M.; Pei, D.; Cheng, C.; Pui, C.H.; Relling, M.V.; e al.
Gene-exp ession pa e ns in d ug- esis an acu e lymphoblas ic leukemia cells and esponse o ea men . N. Engl. J. Med.
2004
,351,
533–542. [C ossRe ]
38.
S ams, W.A.; den Boe , M.L.; Be e loo, H.B.; Meije ink, J.P.; S ig e , R.L.; an We ing, E.R.; Janka-Schaub, G.E.; Sla e , R.; Pie e s, R.
Sensi i i y o L-aspa aginase is no associa ed wi h exp ession le els o aspa agine syn he ase in (12;21)+ pedia ic ALL. Blood
2003,101, 2743–2747. [C ossRe ]
39.
Appel, I.M.; den Boe , M.L.; Meije ink, J.P.; Vee man, A.J.; Renie s, N.C.; Pie e s, R. Up- egula ion o aspa agine syn he ase
exp ession is no linked o he clinical esponse L-aspa aginase in pedia ic acu e lymphoblas ic leukemia. Blood
2006
,107,
4244–4249. [C ossRe ] [PubMed]
40.
Dong, J.; Qiu, H.; Ga cia-Ba io, M.; Ande son, J.; Hinnebusch, A.G. Uncha ged RNA ac i a es GCN2 by displacing he p o ein
kinase moie y om a bipa i e RNA-binding domain. Mol. Cell 2000,6, 269–279. [C ossRe ]
41.
Nakamu a, A.; Nambu, T.; Eba a, S.; Hasegawa, Y.; Toyoshima, K.; Tsuchiya, Y.; Tomi a, D.; Fujimo o, J.; Ku asawa, O.;
Takaha a, C.; e al. Inhibi ion o GCN2 sensi izes ASNS-low cance cells o aspa aginase by dis up ing he amino acid esponse.
P oc. Na l. Acad. Sci. USA 2018,115, E7776–E7785. [C ossRe ]
42.
Wo el, I.M.N.; an de Mee , L.T.; Kilbe g, M.S.; an Leeuwen, F.N. Su i ing S ess: Modula ion o ATF4-Media ed S ess
Responses in No mal and Malignan Cells. T ends Endoc inol. Me ab. 2017,28, 794–806. [C ossRe ]
43.
Qing, G.; Li, B.; Vu, A.; Skuli, N.; Wal on, Z.E.; Liu, X.; Mayes, P.A.; Wise, D.R.; Thompson, C.B.; Ma is, J.M.; e al. ATF4 egula es
MYC-media ed neu oblas oma cell dea h upon glu amine dep i a ion. Cance Cell 2012,22, 631–644. [C ossRe ]
44.
Zinszne , H.; Ku oda, M.; Wang, X.; Ba ch a o a, N.; Ligh oo , R.T.; Remo i, H.; S e ens, J.L.; Ron, D. CHOP is implica ed in
p og ammed cell dea h in esponse o impai ed unc ion o he endoplasmic e iculum. Genes De .
1998
,12, 982–995. [C ossRe ]
45.
Oyadoma i, S.; Koizumi, A.; Takeda, K.; Go oh, T.; Aki a, S.; A aki, E.; Mo i, M. Ta ge ed dis up ion o he Chop gene delays
endoplasmic e iculum s ess-media ed diabe es. J. Clin. In es ig. 2002,109, 525–532. [C ossRe ]
46.
Choi, W.H.; de Poo , S.A.; Lee, J.H.; Kim, J.H.; Han, D.H.; Kim, Y.K.; Finley, D.; Lee, M.J. Open-ga e mu an s o he mammalian
p o easome show enhanced ubiqui in-conjuga e deg ada ion. Na . Commun. 2016,7, 10963. [C ossRe ] [PubMed]
47.
Guima aes, J.C.; Za olan, M. Pa e ns o ibosomal p o ein exp ession speci y no mal and malignan human cells. Genome Biol.
2016,17, 236. [C ossRe ] [PubMed]
48.
Shalem, O.; Sanjana, N.E.; Ha enian, E.; Shi, X.; Sco , D.A.; Mikkelson, T.; Heckl, D.; Ebe , B.L.; Roo , D.E.; Doench, J.G.; e al.
Genome-scale CRISPR-Cas9 knockou sc eening in human cells. Science 2014,343, 84–87. [C ossRe ] [PubMed]
49.
Xiong, X.; Zhao, Y.; He, H.; Sun, Y. Ribosomal p o ein S27-like and S27 in e play wi h p53-MDM2 axis as a a ge , a subs a e and
a egula o . Oncogene 2011,30, 1798–1811. [C ossRe ]
In . J. Mol. Sci. 2023,24, 13260 18 o 18
50.
Bai, D.; Zhang, J.; Xiao, W.; Zheng, X. Regula ion o he HDM2-p53 pa hway by ibosomal p o ein L6 in esponse o ibosomal
s ess. Nucleic Acids Res. 2014,42, 1799–1811. [C ossRe ] [PubMed]
51.
Vabulas, R.M.; Ha l, F.U. P o ein syn hesis upon acu e nu ien es ic ion elies on p o easome unc ion. Science
2005
,310,
1960–1963. [C ossRe ] [PubMed]
52.
Su awee a, A.; Münch, C.; Hanssum, A.; Be olo i, A. Failu e o amino acid homeos asis causes cell dea h ollowing p o easome
inhibi ion. Mol. Cell 2012,48, 242–253. [C ossRe ] [PubMed]
53.
Linds öm, M.S.; Zhang, Y. Ribosomal p o ein S9 is a no el B23/NPM-binding p o ein equi ed o no mal cell p oli e a ion.
J. Biol. Chem. 2008,283, 15568–15576. [C ossRe ] [PubMed]
54.
Oli e , E.R.; Saunde s, T.L.; Ta lé, S.A.; Glase , T. Ribosomal p o ein L24 de ec in belly spo and ail (Bs ), a mouse Minu e.
De elopmen 2004,131, 3907–3920. [C ossRe ]
55.
Mazumde , B.; Sampa h, P.; Seshad i, V.; Mai a, R.K.; DiCo le o, P.E.; Fox, P.L. Regula ed elease o L13a om he 60S ibosomal
subuni as a mechanism o ansc ip -speci ic ansla ional con ol. Cell 2003,115, 187–198. [C ossRe ]
56.
Ho os, R.; Ijspee , H.; Pospisilo a, D.; Send ne , R.; And ieu-Sole , C.; Taskesen, E.; Nie adka, A.; Cmejla, R.; Send ne , M.;
Touw, I.P.; e al. Ribosomal de iciencies in Diamond-Black an anemia impai ansla ion o ansc ip s essen ial o di e en ia ion
o mu ine and human e y h oblas s. Blood 2012,119, 262–272. [C ossRe ]
57.
Kond asho , N.; Pusic, A.; S ump , C.R.; Shimizu, K.; Hsieh, A.C.; Ishijima, J.; Shi oishi, T.; Ba na, M. Ribosome-media ed
speci ici y in Hox mRNA ansla ion and e eb a e issue pa e ning. Cell 2011,145, 383–397. [C ossRe ]
58. Sulima, S.O.; Kampen, K.R.; De Kee smaecke , K. Cance Biogenesis in Ribosomopa hies. Cells 2019,8, 229. [C ossRe ]
59. Na la, A.; Ebe , B.L. Ribosomopa hies: Human diso de s o ibosome dys unc ion. Blood 2010,115, 3196–3205. [C ossRe ]
60.
Hannan, K.M.; Sanij, E.; Ro hblum, L.I.; Hannan, R.D.; Pea son, R.B. Dys egula ion o RNA polyme ase I ansc ip ion du ing
disease. Biochim. Biophys. Ac a 2013,1829, 342–360. [C ossRe ]
61.
Aspesi, A.; Ellis, S.R. Ra e ibosomopa hies: Insigh s in o mechanisms o cance . Na . Re . Cance
2019
,19, 228–238. [C ossRe ]
[PubMed]
62.
Dameshek, W. Riddle: Wha do aplas ic anemia, pa oxysmal noc u nal hemoglobinu ia (PNH) and “hypoplas ic” leukemia ha e
in common? Blood 1967,30, 251–254. [C ossRe ] [PubMed]
63. Mills, E.W.; G een, R. Ribosomopa hies: The e’s s eng h in numbe s. Science 2017,358, eaan2755. [C ossRe ]
64.
Gue a-Mo eno, A.; Isasa, M.; Bhanu, M.K.; Wa e man, D.P.; Eapen, V.V.; Gygi, S.P.; Hanna, J. P o eomic Analysis Iden i ies
Ribosome Reduc ion as an E ec i e P o eo oxic S ess Response. J. Biol. Chem. 2015,290, 29695–29706. [C ossRe ]
65.
Albe , B.; Kos-B aun, I.C.; Hen as, A.K.; Dez, C.; Rueda, M.P.; Zhang, X.; Gadal, O.; Kos, M.; Sho e, D. A ibosome assembly
s ess esponse egula es ansc ip ion o main ain p o eome homeos asis. eLi e 2019,8, e45002. [C ossRe ]
66.
Doench, J.G.; Fusi, N.; Sullende , M.; Hegde, M.; Vaimbe g, E.W.; Dono an, K.F.; Smi h, I.; To ho a, Z.; Wilen, C.; O cha d, R.; e al.
Op imized sgRNA design o maximize ac i i y and minimize o - a ge e ec s o CRISPR-Cas9. Na . Bio echnol.
2016
,34, 184–191.
[C ossRe ]
67.
Sanson, K.R.; Hanna, R.E.; Hegde, M.; Dono an, K.F.; S and, C.; Sullende , M.E.; Vaimbe g, E.W.; Goodale, A.; Roo , D.E.;
Piccioni, F.; e al. Op imized lib a ies o CRISPR-Cas9 gene ic sc eens wi h mul iple modali ies. Na . Commun.
2018
,9, 5416.
[C ossRe ]
68.
Sadelain, M.; Papape ou, E.P.; Bushman, F.D. Sa e ha bou s o he in eg a ion o new DNA in he human genome. Na . Re .
Cance 2011,12, 51–58. [C ossRe ]
69.
A iës, I.M.; Bodaa , K.; Ka im, S.A.; Chonghaile, T.N.; Hinze, L.; Bu ns, M.A.; P i mann, M.; Dega , J.; Land igan, J.T.;
Balbach, S.; e al. PRC2 loss induces chemo esis ance by ep essing apop osis in T cell acu e lymphoblas ic leukemia. J. Exp. Med.
2018,215, 3094–3114. [C ossRe ]
70.
Bushnell, B. BBMap: A Fas , Accu a e, Splice-Awa e Aligne . In P oceedings o he 9 h Annual Genomics o Ene gy & En i onmen
Mee ing, Walnu C eek, CA, USA, 17–20 Ma ch 2014.
71.
Bolge , A.M.; Lohse, M.; Usadel, B. T immoma ic: A lexible imme o Illumina sequence da a. Bioin o ma ics
2014
,30, 2114–2120.
[C ossRe ] [PubMed]
72.
B ay, N.L.; Pimen el, H.; Mels ed, P.; Pach e , L. Nea -op imal p obabilis ic RNA-seq quan i ica ion. Na . Bio echnol.
2016
,34,
525–527. [C ossRe ] [PubMed]
73.
Cunningham, F.; Allen, J.E.; Allen, J.; Al a ez-Ja e a, J.; Amode, M.R.; A mean, I.M.; Aus ine-O imoloye, O.; Azo , A.G.; Ba nes,
I.; Benne , R.; e al. Ensembl 2022. Nucleic Acids Res. 2021,50, D988–D995. [C ossRe ] [PubMed]
74.
Ha , T.; Komo i, H.K.; LaMe e, S.; Podshi alo a, K.; Salomon, D.R. Finding he ac i e genes in deep RNA-seq gene exp ession
s udies. BMC Genom. 2013,14, 778. [C ossRe ]
75.
Li, W.; Xu, H.; Xiao, T.; Cong, L.; Lo e, M.I.; Zhang, F.; I iza y, R.A.; Liu, J.S.; B own, M.; Liu, X.S. MAGeCK enables obus
iden i ica ion o essen ial genes om genome-scale CRISPR/Cas9 knockou sc eens. Genome Biol.
2014
,15, 554. [C ossRe ]
[PubMed]
Disclaime /Publishe ’s No e:
The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual
au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o
people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .