GSK3α Regulates Temporally Dynamic Changes in Ribosomal Proteins upon Amino Acid Starvation in Cancer Cells

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. 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