Targeting Oncogenic NOTCH1 in T-cell Acute Lymphoblastic Leukemia With a Selective SERCA Inhibitor CAD204520

UNIVERSITA’ DEGLI STUDI DI PARMA DOTTORATO DI RICERCA IN “SCIENZE MEDICHE E CHIRUGICHE TRASLAZIONALI” CICLO XXXV TARGETING ONCOGENIC NOTCH1 IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA WITH A SELECTIVE SERCA INHIBITOR CAD204520 Coo dina o e: Chia .mo P o . Nicola S e zella i Tu o e: Chia .mo P o . Gio anni Ro i Co-Tu o e: Chia .mo P o . Pie e Van Vlie be ghe Do o ando: D . Luca Paglia o Anni Accademici 2019/2020 – 2021/2022 1 ABSTRACT The iden i ica ion o SERCA (sa co/endoplasmic e iculum calcium ATPase) as a a ge o modula ing gain-o - unc ion NOTCH1 mu a ions in No ch dependen cance s has spu ed he de elopmen o his compound class o cance he apeu ics. SERCA plays a c i ical ole in Ca2+ egula ion pa icula ly in myocy es, hus inhibi ing i may inc ease he isk o hea ailu e, limi ing he de elopmen o his compound class o cance he apeu ics. Conside ing his oxici y challenge, disco e y and ea ly op imiza ion o small molecules wi h be e d ug- like p ope ies and educed o - a ge oxici y is wa an ed. Despi e he inna e oxici y challenge associa ed wi h SERCA inhibi ion, we iden i ied CAD204520, a small molecule wi h be e d ug-like p ope ies and educed o - a ge Ca2+ oxici y compa ed wi h he SERCA inhibi o hapsiga gin. Simila o he SERCA inhibi o hapsiga gin, CAD204520 inhibi s No ch signaling in T-ALL leukemia cells causing a cell cycle a es and induc ion o apop osis. CAD204520 also p e e en ially a ge s mu a ed o e wild ype NOTCH1 p o eins in T cell acu e lymphoblas ic leukemia (T-ALL) and man le cell lymphoma (MCL), he eby o e coming he he apeu ic limi a ion associa ed wi h he use o pan-No ch inhibi o s. Rema kably, CAD204520 esul ed in an e ec i e ea men in a NOTCH1-mu a ed T-ALL in i o model wi hou causing o e Ca2+- ela ed ca diac oxici y. To an icipa e he po en ial mechanism o esis ance o SERCA inhibi o s, we gene a ed a T-ALL cell line esis an o he e ec o hapsiga gin. A subsequen small molecule lib a y sc eening iden i ied glucoco icoids among he op hi s in he esis an cell line. This e ec is a leas in pa media ed by he speci ic up egula ion o glucoco icoid ecep o . Consequen ly, he associa ion o SERCA inhibi o s and glucoco icoids displayed a syne gis ic e ec in mul iple p eclinical models. This s udy suppo s he de elopmen o SERCA inhibi o s o No ch- dependen cance s and ex ends hei applica ion o cases wi h isola ed mu a ions in he 2 PEST deg ada ion domain o NOTCH1, such as MCL o ch onic lymphocy ic leukemia (CLL). Fu he mo e, his s udy sugges s ha SERCA-Ca2+ modula ion media es glucoco icoid signaling and ha inno a i e SI can pha macologically modula e glucoco icoid esis ance in T-ALL. 3 INDEX INTRODUCTION .................................................................................................................. 5 Ta ge ing No ch T a icking ............................................................................................... 5 SERCA ............................................................................................................................. 5 SERCA and Cance ........................................................................................................ 11 SERCA and No ch .......................................................................................................... 13 AIM OF THE STUDY ......................................................................................................... 17 MATERIALS AND METHODS ........................................................................................... 19 Expe imen al Models and Subjec De ails ...................................................................... 19 Da a and Code A ailabili y .............................................................................................. 21 P epa a ion, C ys alliza ion and S uc u e De e mina ion o he SERCA-CAD204520 Complex .......................................................................................................................... 22 Syn hesis Pa hways ........................................................................................................ 23 ATPase P epa a ion ....................................................................................................... 31 ATP Hyd olysis Inhibi ion ................................................................................................ 32 Cell Viabili y, Apop osis and DNA Con en Assays ......................................................... 33 Cell Compe i ion Assay ................................................................................................... 34 Compound Sou ces ........................................................................................................ 34 Compound T ea men o Cell Lines and P ima y Cells ................................................... 34 In acellula Calcium Measu emen ................................................................................ 35 Wes e n Blo ................................................................................................................... 36 Indi ec Immuno luo escence Mic oscopy ...................................................................... 37 Real- ime RT-PCR .......................................................................................................... 38 Whole Exome Sequencing ............................................................................................. 38 Vi us P oduc ion and T ansduc ion o T-ALL Cell Lines ................................................. 39 Ca diomyocy e Isola ion and T ea men ......................................................................... 40 In acellula ATP Con en De ec ion ............................................................................... 42 In Vi o S udies ................................................................................................................ 42 Small molecule sc eening assay .................................................................................... 44 Quan i ica ion and S a is ical Analysis ............................................................................ 44 RESULTS ........................................................................................................................... 45 4 Iden i ica ion o CAD204520 as a Selec i e Ca2+ ATPase Inhibi o ................................ 45 CAD204520 Rescues T-ALL Cells om Thapsiga gin Resis ance ................................. 48 CAD204520 Supp esses Leukemia G ow h in NOTCH1-Mu a ed T-ALL and MCL ....... 51 CAD204520 Supp esses No ch1 Signaling .................................................................... 51 CAD204520 P e e en ially Inhibi s NOTCH1-Mu a ed Cance s ..................................... 53 Consequences o Ca2+ Release upon CAD204520 T ea men ...................................... 54 Modeling P eclinical Toxici y and E icacy o CAD204520 in a T-ALL Leukemia Model 56 Iden i ica ion o glucoco icoids as po en ial escue ea men in NOTCH1-mu a ed cell line ca ying ATP2A2 mu a ion ....................................................................................... 59 ATP2A2 mu a ion induces up egula ion o glucoco icoid ecep o ................................ 60 SERCA inhibi o s syne gize wi h glucoco icoids wi h a be e p o ile in ATP2A2 mu a ed cells ................................................................................................................................. 61 DISCUSSION ..................................................................................................................... 62 CONCLUSIONS ................................................................................................................. 68 BIBLIOGRAPHY ................................................................................................................ 70 FIGURE LEGENDS ........................................................................................................... 89 5 INTRODUCTION Ta ge ing No ch T a icking The No ch signaling pa hway plays an impo an ole in he pa hogenesis o human cance s. No ch con ols bo h cell-in insic and ex insic ci cui s leading o umo de elopmen , p og ession and esponse o he apy. Se e al he apeu ic e o s ha e his o ically ocused on modula ing No ch signaling by using small molecules such as g-sec e ase inhibi o s (GSI) o an ibody-based s a egies. Howe e , hese app oaches ha e a poo he apeu ic window - wild ype s mu an p o eins - limi ing hei applica ion in human diseases. This is no he case o small molecules a ge ing he sa co-endoplasmic e iculum Ca2+-ATPase (SERCA). SERCA inhibi ion hijacks No ch a icking and i s ac i a ion, eme ging as d uggable app oach o NOTCH-dependen cance s. SERCA SERCA p o eins belong o he supe amily o ac i e anspo e s known as P- ype ATPases (E1/E2- ype) loca ed in he endoplasmic e iculum (ER). In 1993 Toyoshima and colleagues desc ibed he i s comple e s uc u e o SERCA by c yo-elec on mic oscopy (1). Subsequen ly, no el high- esolu ion echniques shaped he esolu ion o se e al c ys allog aphy s uc u es o SERCA. These s udies showed how ligands (e.g., anada e, hapsiga gin) bind SERCA, and wha s uc u al changes occu du ing he enzyma ic ca aly ic cycle (2-6). The SERCA p o ein comp ises en helices (M1-M10), a small luminal ail and h ee cy oplasmic domains (A, ac ua o ; N, nucleo ide binding; P, phospho yla ion). These modules media e ATP hyd olysis, hyd on (H+) and calcium (Ca2+) binding and hei anspo h ough ER memb ane (7). ATP2A1 (16p11.2), ATP2A2 (12q24.11) and ATP2A3 (17p13.2) genes encode o SERCA1, 2 and 3 espec i ely (8, 9). Today, o e 70 SERCA iso o ms 6 esul ing om al e na i e splicing a e deposi ed in he P o ein Da a Bank da abase (www. csb.o g). While hese ansc ip s sha e up o 85% o sequence homology, di e ences in issue dis ibu ion, Ca2+ binding a ini y bo h in no mal and cance issue a e due o changes in he p o ein C- e minal egion (10-15). SERCA p o eins main ain in acellula Ca2+ homeos asis by pumping Ca2+ om cy osol in o he ER (16). This adenosine iphospha e (ATP) (10) dependen ca aly ic cycle al e na es di e en SERCA phospho yla ed in e media es (E1P; E2P) wi h di e en con o ma ion o high (E1-Mic omola -μM) o low (E2-Millimola -mM) a ini y o Ca2+: his cycle, named Pos - Albe s scheme by he i s p oponen , was o iginally applied o o he ypes o ion pump, such as he Na+/K+-ATPase, and subsequen ly adap ed o SERCA (17). The ne balance o he ATP hyd olysis equi ed o comple e he cycle is o wo Ca2+ ions ans e ed om he cy osol o he ER lumen, and wo o h ee p o ons in he opposi e di ec ion owa ds he cy osol (18) (Figu e 1). All SERCA iso o ms p esen wo Ca2+ binding si es (si e I and II) be ween ou ansmemb ane (TM) helices (M4, M5, M6 and M8), nea he cy oplasmic side o he memb ane. Si e I con ains i e amino acids esidues om h ee di e en ansmemb ane helices associa ed wi h wo wa e molecules: Asn768 and Glu771 om M5, Th 799 and Asp800 om M6 and Glu908 om M8 (19). Si e II is loca ed on he cy oplasmic su ace nex o he si e I and i is composed o Ile307 and Gly310 om M4 helix, ha is pa ially unwound, and Asn796 and Asp800 om M6; bo h si es p o ide one o h ee chain o oxygen o he co- o dina ion (19). In e es ingly, he binding o he i s Ca2+ ion on si e I ac s in a coope a i e way, b inging o a con o ma ional change on he oxygen chains o si e II and allowing an high a ini y s a e o he second ion binding (16, 19, 20). 7 SERCA, is o ganized in unc ional domains ha media e ions binding, ATP hyd olysis and ions anspo h ough memb ane (7). The N domain is he la ges one o accommoda e h ough he binding a he Phe487 esidue, he adenine ing o he ATP (19, 21, 22). The N- domain is linked, h ough he A g560 esidue o he su ounding enzyma ic P domain ha con ains he amino acid esidues a ge ed by he phospho yla ion. Fo example, phospho yla ion o he Asp351 esidue allows o a 25-30 Å ea angemen s o he P-domain- ATP complex leading o gene a ion o he high ene gy enzyme in e media e (16, 20, 22, 23). The A domain ealizes he ga ing mechanism o calcium anspo . The A domain is he smalles cy osolic domain and is si ua ed nea he M1, M2 and M3 helices wi h which is bound by lexible linke s allowing a be e ange o mo emen o ATP u iliza ion. Simila ly o o he P- ype ATPases, he A domain p esen s conse ed p o ein mo i es such as he TGES mo i , esponsible o he dephospho yla ion o he Asp351 esidue du ing he ca aly ic cycle (16, 19, 20, 23, 24). The high- esolu ion c ys al s uc u es o he SERCA bound o Ca2+, hapsiga gin, and anada e p o ided he ame o he unde s anding o s uc u al changes occu ing du ing he ca aly ic cycle (19). Thapsiga gin binds o SERCA i ing in a ca i y composed by M3, M5 and M7 helices (25). The mos impo an amino acid esidue ha in e ac s wi h Thapsiga gin is Phe256 on M3 helix, assis ed by Ile765 on M5 and Ty 837 on M7 (25, 26). Mu a ions a ec ing he esidue Phe256 b ing o hapsiga gin esis ance con i ming he pi o al ole o his amino acid in he inhibi ion o he enzyme, e en i he educ ion o sensi i i y o SERCA caused by he mu a ion is di e en among he iso o ms, wi h SERCA2b he leas a ec ed (14, 26). O he SERCA inhibi o s, like cyclopiazonic acid (CPA) o 2,5-di-( -bu yl)-1,4-benzohyd oquinone (DBHQ), bind he enzyme in a di e en pocke , be ween he ansmemb ane helices M1, M2, M3 and M4 (27, 28), iden i ied as he en ance pa hway o calcium ions om cy osol (29). 14 and in a subse o B-cell-like (ac i a ed B-cell, ABC) di use la ge B-cell lymphoma (94). Mos o hese mu a ions occu in he jux amemb ane he e odime iza ion (HD) domain, which hold oge he he ECD-N wi h TM-N, o in he PEST ( ich in p oline (P), glu amic acid (E), se ine (S), and h eonine (T)) deg on domain. In T-ALL, HD and PEST mu a ions may co- occu while isola ed PEST a e common in CLL and MCL. He e, a p ema u e s op codon (P2514 s*4) gene ally unca es he C- e minal PEST egion o he p o ein. The PEST domain con ols he deg ada ion o ac i a ed NOTCH p o eins, and i s dele ion esul s in a delayed p o ein hal -li e. In addi ion, he No ch signaling pa hway is equen ly ac i a ed in mul iple ypes o solid umo s (95, 96), such as melanoma, colo ec al ca cinoma, and cholangioca cinoma h ough mechanisms ha di e om gene ic a ia ions (97, 98). Pa adoxically, mu a ions ha inac i a e he No ch pa hway ha e been desc ibed in se e al human cance s (96, 99) showing ha , depending on he cellula con ex , No ch signaling can be oncogenic o umo supp essi e and sugges ing ha ine- uned inhibi ion o No ch signaling could be use ul in hose si ua ions whe e No ch is ac i a ed. The p eponde ance o oncogenic NOTCH1 mu a ions in T-ALL has p omp ed he sea ch o e ec i e an i-No ch1 he apeu ics (75, 100). Because No ch ac i a ion elies on g-sec e ase media ed p o eolysis, GSI had en e ed in clinical ials o ea elapsed T-ALL. Howe e , i s gene a ions o GSIs we e poo ly ole a ed because o on- a ge gas o-in es inal oxici y (101-103). As showed by Riccio and colleagues he oxic e ec o GSIs a e a consequence o lack o subs a e speci ici y o hese molecules esul ing in he combined inhibi ion o wild ype NOTCH1 and NOTCH2 in in es inal p ogeni o cells (104). Al hough ew pa ien s achie ed a comple e esponse, GSIs exhibi ed mode a e clinical ac i i y in some pa ien s wi h solid umo and leukemia (105). Recen ly, se e al s udies demons a ed ha combining 15 GSIs wi h chemo he apy o o he a ge ed agen s inc eases he an i-cance e ec s o hese d ugs (106-109), suppo ing he de elopmen o inno a i e an i-No ch1 he apeu ics. The ise o SERCA inhibi o s o cance he apeu ics da e back o 1960 when he Na ional Cance Ins i u e (NCI) launched a p og am o iden i y compounds wi h an i umo ac i i y om 35,000 plan s ex ac s (110). Sesqui e pene lac one (SL) de i a i es demons a ed an an i- in lamma o y and an i umo ac i i y in se e al umo ypes, like la yngeal ca cinomas, u eal melanomas, pi ui a y mac oadenomas, kidney, p os a e cance and hema ological malignancies (111-116). Among o he s, hapsiga gin, pa henolide and a emisinin we e selec ed o hei po ency and ini ially used as ool compounds in di e en cance models. Se e al SERCA inhibi o s which di e ed in hei sou ce, chemical s uc u e, po ency and binding a ini y o speci ic SERCA iso o ms we e subsequen ly de eloped (82). In 1999, Go an Pe iz and Ma k E. Fo ini desc ibed ha he a icking e en s leading o a co ec NOTCH ac i a ion may be dis up ed in he p esence o a de ec i e Ca2+-ATPase unc ion in a D osophila model. In his wo k, he au ho s demons a ed ha loss-o - unc ion alleles o he D osophila SERCA homologous gene Ca-P60A al e s p ope syn hesis, olding and a icking o he NOTCH ecep o in he ER/Golgi compa men s (Figu e 2). Consis en ly, in D osophila S2 cul u ed lines, he ea men wi h gene al SERCA inhibi o s such as hapsiga gin and CPA p ima ily educes he amoun o NOTCH p o eins ha each he cell su ace (117). While ex emely in e es ing, hese obse a ions we e no con i med in mammalian cells un il, Gio anni Ro i and Kimbe ly S egmaie emba ked on a la ge gene- exp ession based sc eening (GE-HTS) e o o iden i y inhibi o s o oncogenic NOTCH1 signa u es o enhance o NOTCH1 HD mu an L1601P∆P ac i i y in T-ALL. Among he op hi s we e he genes ATP2A2 and ATP2A3, and SERCA inhibi o s such hapsiga icin (an analogue o hapsiga gin) and CPA. Toge he wi h o he ion lux modula o s, SERCA 16 eme ged as a no el po en ial he apeu ic a ge in NOTCH1-associa ed cance s (75, 118). Fu he mo e, hese da a sugges he hypo hesis ha No ch signaling could be dys unc ional in se e al gene ic diso de s associa ed wi h loss o unc ion ATPA1-3 mu a ions. Howe e , hapsiga gin binding o SERCA esul s in an inc ease in cy osolic Ca2+ concen a ion and a deple ion o Ca2+ s o ed in he ER. Thus, he deli e y o ee hapsiga gin o humans migh cause ca diac oxici y due o a apid calcium ion shi . To o e come his limi a ion in he pas we gene a ed a hapsiga gin p o-d ug aking ad an age o he dependency o ALL on olic acid (FA) and, agged ola e o a pe missi e si e on an ac i e alcohol de i a i e o hapsiga gin (8-O-debu anoyl hapsiga gin) ia a clea able es e linkage (JQ-FT) (119). Howe e , an al e na i e app oach o educing he po en ial oxici y o hapsiga gin is h ough he iden i ica ion o SERCA inhibi o s ha e ain he an i-No ch p ope ies bu lack Ca2+ ela ed oxici ies (Figu e 3). 17 AIM OF THE STUDY Gi en i s c i ical oncogenic ole in se e al human cance s, No ch1 signaling has ga ne ed inc eased a en ion as a po en ial he apeu ic a ge . To da e, se e al No ch inhibi o s, including GSI, ha e shown he apeu ic e icacy in p eclinical cance models. Howe e , despi e his p omise, ew o hese candida es ha e been demons a ed o ha e a meaning ul clinical bene i o pa ien s, in pa due o issue-dependen on- a ge oxici ies om he simul aneous ep ession o bo h mu an and wild ype NOTCH p o eins. The disco e y o SERCA as ac ionable modula o s o No ch1 sugges ed a new a ge ed app oach o ea T-ALL. Uniquely among No ch modula o s, SERCA inhibi o s p e e en ially impai he clinically ele an class o oncogenic NOTCH1 mu an s compa ed o wild ype. Thus, he de elopmen o ole a ed SERCA modula o s may unco e a new he apeu ic a enue o one o he mos equen ly mu a ed genes in human cance s. In his wo k, we iden i ied a new se ies o P- ype ATPase/SERCA inhibi o s and cha ac e ized he e ec o CAD204520 in NOTCH1-mu a ed T-ALL. We demons a ed ha CAD204520 exhibi s a educed Ca2+ ela ed o - a ge oxici y bu e ains an i-No ch1 and an i-leukemia capaci y bo h in i o and in i o in NOTCH1-mu a ed T-ALL models. In addi ion, we ex ended he es ing o CAD204520 in B-cell malignancies ca ying clinically ele an PEST mu a ions and demons a ed, o he i s ime, he po en ial o SERCA inhibi ion as a he apeu ic app oach in hese diseases. Fu he mo e, o an icipa e he po en ial mechanism o esis ance o SERCA inhibi o s, we gene a ed a T-ALL cell line (ALL/SIL) esis an (R) o he e ec o hapsiga gin by exposing he cells o inc eased concen a ions o hapsiga gin. Exome sequencing analysis iden i ied a mu a ion (c.G770Tà p.G257V) in he ATP2A2 locus ha impedes an e icien hapsiga gin 18 binding o he p o ein, inally esul ing in a diminished inhibi o y e ec . we hen sc eened a small molecule lib a y o nea ly 2500 bioac i e compounds ( om he Eu opean Chemical Biology Lib a y p o ided by EU-OPENSCREEN) in ALL/SIL and ALL/SIL R. Compound hi s we e lagged by hei abili y o p e e en ially inhibi s ALL/SIL, ALL/SIL R, o bo h. Con i ma o y expe imen s and pa hway analysis we e comple ed in mul iple T-ALL p eclinical models. 19 MATERIALS AND METHODS Expe imen al Models and Subjec De ails Animals NOD-scid IL2Rgammanull (NSG) mice (The Jackson labs, RRID: IMSR_JAX:005557) o e icacy s udies we e main ained in speci ic pa hogen– ee acili ies a he ‘‘P eclinical Resea ch Se ices Cen e ’’ (Ce.Se.R.P) a he Uni e si y o Pe ugia (08/2018-UT o 07/24/2018). Animal p ocedu es we e app o ed by he Uni e si y o Pe ugia IACUC ollowing he DL 26/2014 and 2010/63/EU guidelines o he p o ec ion o animals used o scien i ic pu poses. Pha macokine ics (PK) and ole abili y s udies we e pe o med a Au igene Disco e y Technologies, India. In-house b eed CD1 (ICR) o BALB/cAnNC mice o pha macokine ics (PK) and ole abili y s udies we e main ained in indi idually en ila ed cages a he Au igene acili y in Hyde abad in India. To assess e ec o CAD204520 on he ca diac mechanics, ca diomyocy es we e isola ed om 12-14 week aged Wis a a s (Ra us no egicus, Cha les Ri e Labo a o y, RRID: RGD_13508588) o 362 ± 5 g in weigh . Animals we e housed in a empe a u e-con olled oom (22–24°C), wi h a 12 hou ligh cycle (ligh on om 7.00 AM o 7.00 PM) wi h un es ic ed ood and wa e supply. Expe imen s we e pe o med unde he Ve e ina y Animal Ca e and Use Commi ee o he Uni e si y o Pa ma-I aly and con o m o he Na ional E hical Guidelines o he I alian Minis y o Heal h (P o . N 614/2016-PR) and he Guide o he Ca e and Use o Labo a o y Animals (Na ional Ins i u e o Heal h, Be hesda, MD, USA, e ised 1996). Yeas Cells Saccha omyces ce e isiae RS72 yeas cells (ATCC #9763) o he biochemical ATPase assay we e p e-cul u ed in 100 ml s e ile SGAH medium (7.04 g/L yeas ni ogen base, 19.8 20 g/L galac ose, 64 mg/L adenine, 64 mg/L his idine) o 3 days a 25°C and 150 pm. The p e-cul u e was ans e ed o 500 mL s e ile SGAH medium and u he incuba ed o 3 o 4 days. 100 mL om he cell cul u e was ans e ed o 1 L YPAD medium (10 g/L yeas ex ac , 20 g/L bac o-pep one, 20 g/L glucose, 20 mg/L adenine) and incuba ed a 25°C o 18-20 hou s. Cell Lines Human cell lines DND41 (sou ce: male), MOLT16 (sou ce: emale), PF382 (sou ce: emale), RPMI-8402 (sou ce: emale), SKW-3/KE-37 (sou ce: male), JURKAT (sou ce: male), CTV- 1 (sou ce: male), HBS2 (sou ce: male), Loucy (sou ce: emale) and PEER (sou ce: emale), REC-1 (sou ce: male), Mino (sou ce: emale), we e pu chased om Leibniz-Ins i u DSMZ- Ge man collec ion o mic oo ganisms and cell cul u es (Ge many); he iden i y o ALL/SIL (sou ce: male) was con i med by PCR sequencing o known NOTCH1 mu a ions and sho andem epea (STR) loci p o iling and hey we e kindly p o ided by S egmaie labo a o y. MAVER-1 (sou ce: male) cells we e a gi om he Mu a o labo a o y (C.R.O. Na ional Cance Ins i u e, A iano, I aly). Cells we e cul u ed in RPMI 1640 (Fishe Scien i ic, Wal ham MA, USA #MT10040CV) wi h 10% o 20% e al bo ine se um (FBS) (The mo Fishe Scien i ic, Wal ham MA, USA, #10270-106) and 1% penicillin-s ep omycin (Fishe Scien i ic, Wal ham MA, #3MT30002CI) and incuba ed a 37°C wi h 5% CO2. 293T (sou ce: human p ima y emb yonal kidney cell line 293 (ACC 305)) cells we e pu chased Leibniz- Ins i u DSMZ-Ge man collec ion o mic oo ganisms and cell cul u es (Ge many) and cul u ed in DMEM (Fishe Scien i ic, Wal ham MA, USA #11965-084) wi h 10% FBS and 1% penicillin-s ep omycin and incuba ed a 37°C wi h 5% CO2. HL-1 ca diac muscle cell line was a kind gi om he Mi agoli labo a o y (Uni e si y o Pa ma, Pa ma, I aly). HL-1 cells we e pla ed on a gela in laye de i ed om bo ine skin/ ib onec in (1mg/ml) (Sigma-Ald ich, S . Louis, MO, USA, #G9391 and #F-1141) coa ed T25 lask and cul u ed in Claycomb 21 medium (Sigma-Ald ich, S . Louis, MO, USA, #51800C) wi h 10% FBS, 1% penicillin- s ep omycin, 2mM L-Glu amine (The mo Fishe Scien i ic, Wal ham MA, USA, Wal ham MA, USA, #25030081), 0,1mM No epineph ine [(±)- A e enol] plus L-Asco bic acid, sodium sal (Sigma-Ald ich, S . Louis, MO, USA, #A0937 and #A7506) and incuba ed a 37°C wi h 5% CO2. Cy ogene ic, FISH and mu a ion analysis was comple ed acco ding o alida ed me hods as p e iously desc ibed (120, 121). P ima y Samples T-ALL lymphoblas s we e ob ained om pa ien s wi h leukemia unde an app o ed p o ocol a he Pa ma Uni e si y Hospi al (n.18249/18/05/2017) and acco ding o he decla a ion o Helsinki guidelines o he p o ec ion o human igh s. Pe iphe al blood (PB), and bone ma ow (BM) samples we e collec ed a he ime o diagnosis, and we e ained samples wi h blas s >85%. Mononuclea cells we e isola ed by densi y g adien cen i uga ion using LSM- lymphocy e sepa a ion medium (CappelTM MP Biomedicals, LLC, Ohio, USA #50494). Lymphocy es we e isola ed om pe iphe al blood mononuclea cell (PBMC) by using a CliniMACS P odigy (Mil enyi Bio ec, Be gisch Gladbach, Ge many) and cul u ed o a sho ime using he same g ow h condi ions desc ibed abo e. Da a and Code A ailabili y The model and s uc u e ac o s o he SERCA-CAD204520 complex s uc u e epo ed in his pape ha e been deposi ed in he P o ein Da a Bank: PDB: 6YAA. 22 P epa a ion, C ys alliza ion and S uc u e De e mina ion o he SERCA-CAD204520 Complex Rabbi sa coplasmic e iculum (SR) memb anes con aining SERCA we e p epa ed om abbi hind leg muscle as p e iously desc ibed (122). B ie ly, muscle issue was dissec ed and minced in 10 mM KCl, 2.5 mM K2HPO4, 2.5 mM KH2PO4, 2 mM EDTA, ollowed by cen i uga ion a 4°C o 20 min and 6.400 x g, supe na an il e ed and spun a 9.700 x g o 20 min. SR memb anes we e sedimen ed by a 60 min cen i uga ion a 47.800 x g a 4°C. Memb anes we e homogenised and washed successi ely wi h bu e s B (1 M suc ose, 50 mM KCl), C (1 M KCl, 3.4 mM MgATP pH 7.0), D (50 mM KCl), and E (0.3 M suc ose, 5 mM Hepes pH 7.4). Finally, washed memb anes we e ex ac ed wice wi h ex ac ion bu e (0.3 M suc ose, 0.5 M KCl, 1 mM EDTA, 10 mM T is, 0.01 mM CaCl2, 1.25 mM MgCl2, pH 7.9, 0.5 mg/ml DOC, 0.5 mg/ml DTT) ollowed by cen i uga ion a 4°C and 181.000 x g o 75 min. The pelle was hen washed wi h 5 mM TAPS pH 7.5, 0.3 M suc ose, 0.5 M KCl, 0.5 mM MgCl2, 10 µM CaCl2, and inally esuspended and lash ozen in bu e E. SERCA memb anes we e esuspended and gen ly homogenized in 100 mM MOPS-T is pH 6.8, 80 mM KCl, 3 mM MgCl2, 4 mM EGTA and 20% ( / ) glyce ol. CAD204520 was added o he memb ane p epa a ion a a inal concen a ion o 0.5 mM and incuba ed o e nigh a 4°C. The ollowing day, 0.4 mM TNPATP we e added and incuba ed o 15 minu es (min) p io o he solubiliza ion o he p o ein wi h C12E8 a a de e gen /p o ein a io o 1.5:1 (w/w). A e 10 min incuba ion and cen i uga ion (TLA-100.3 o o , 50,000 pm, 30 min, 4°C), he concen a ion o solubilized p o ein was usually 10–12 mg/mL. Co-c ys alliza ion o SERCA wi h CAD204520 was ca ied ou using hanging d op equilib a ion a oom empe a u e (RT) wi h p o ein/bu e in a 1:1 a io. The bes di ac ing c ys als we e ob ained wi h c ys alliza ion bu e consis ing o 10% glyce ol, 14% PEG 6000, 100 mM NaCl and 6% MPD. Da a we e collec ed a 100 K and a wa eleng h o 0.976 A a beam line I03 a he 23 Diamond Ligh Sou ce (DLS) in Didco , UK. The da a we e p ocessed using XDS (123) and AIMLESS (124) and he s uc u e was de e mined by molecula eplacemen in PHASER (125) using a SERCA c ys al s uc u e wi h ma ching space g oup (pdb: 4UU0) (126). PHENIX (127) was used o e inemen , ligand i ing and model alida ion and COOT o model building (128). Figu es we e p epa ed wi h Pymol (Molecula G aphics Sys em, Ve sion 2.0 Sch o  dinge , LLC). Syn hesis Pa hways CAD307496 (2-[1-[3-(3-py idyl)p opyl]-2-pipe idyl]-6-( i luo ome hoxy)-1H-indole) was p epa ed h ough he ollowing in e media es: a) In e media e 2-[2-(2-py idyl)e hynyl]-5-( i luo ome hoxy)aniline: 2-B omo-5- ( i luo ome hoxy)aniline (6.21g, 24,2 mM), 2-e hynylpy idine (2.50g, 24.2 mM) and po assium ca bona e (8.38g, 60.6 mM) in NMP (50 mL) we e degassed wi h a gon. Pd(D BPF)Cl2 (474mg, 0.73 mM ) was added and he eac ion hea ed unde a gon o 120°C o 1 hou 45 min. The eac ion cooled and dilu ed wi h wa e , ex ac ed wi h 1:1 cHexane/E OAc (3x 150 ml), washing each ex ac well wi h wa e . The combined ex ac s we e d ied, e apo a ed, and columned on 100 g SNAP ca idge elu ing wi h 0-60% E OAc/cHexane, using 20 column olumes o yield 750 mg o i le compound as a b own solid. This was used wi hou u he pu i ica ion. b) In e media e 2-(2-py idyl)-6-( i luo ome hoxy)-1H-indole: To 2-[2-(2-py idyl)e hynyl]- 5-( i luo ome hoxy)aniline (950mg, 3.41 mM) was added po assium 2-me hylp opan- 2-ola e (383.13 mg, 3.41 mM) in DMF (50 mL) and he mix u e was s i ed a RT o e nigh . The eac ion was quenched wi h 0.4 mL HOAc concen a ed in acuo, dissol ed in die hyl e he and washed wi h sa . NaHCO3 and wa e . The o ganic 30 b) In e media e 2-[2-(2-pipe idyl)-6-( i luo ome hoxy)-1H-indol-3-yl]e hanol was p epa ed simila o in e media e B (s ep (b) o CAD204631). Yield 45%. c) In e media e 2-[2-[1-[(4-b omophenyl)me hyl]-2-pipe idyl]-6-( i luo ome hoxy)-1H- indol-3-yl]e hanol was ob ained using In e media e 2-[2-(2-pipe idyl)-6- ( i luo ome hoxy)-1H-indol-3-yl]e hanol and comme cially a ailable 4- b omobenzaldehyde (CAS 1122-91-4) and a p ocedu e simila o CAD204521. Yield 87%. QC-LCMS (ESI): (m/z) (M+H)+ = 497.1 (M-H)- = 495.1. CAD204630 (2-[1-[(4-b omophenyl)me hyl]-2-pipe idyl]-3-isopen yl-6-( i luo ome hoxy)-1H-indole hyd ochlo ide): a) In e media e 3-isopen yl-2-(2-py idyl)-6-( i luo ome hoxy)-1H-indole: By a me hod simila o ha o CAD204631 s ep (a) abo e using comme cially a ailable 2-B omo- 5-( i luo ome hoxy)aniline (CAS 887267-47-2) and 2-(5-me hylhex-1-ynyl)py idine. Yield 60%. b) In e media e 3-isopen yl-2-(2-pipe idyl)-6-( i luo ome hoxy)-1H-indole: By a me hod simila o in e media e B (s ep b o CAD204631). Yield 56%. c) 2-[1-[(4-b omophenyl)me hyl]-2-pipe idyl]-3-isopen yl-6-( i luo ome hoxy)-1H-indole hyd ochlo ide: By a me hod simila ly o ha o CAD204521 employing comme cially a ailable 4-b omobenzaldehyde (CAS 1122-91-4). Yield 62%. QC-LCMS (ESI): (m/z) (M+H)+ = 523.1 (M-H)- = 521.1. No e: Many o he compounds a e acema es i.e. o enan iome s o dias e eome s. The pu e enan iome was no isola ed 31 ATPase P epa a ion Hea compe en Saccha omyces ce e isiae RS72 yeas cells (129) we e ans o med using a li hium ace a e, single-s anded ca ie DNA/polye hylene glycol me hod and wi h a yeas mul icopy ec o (130) con aining he ull-leng h cDNA o he S. ce e isiae plasma memb ane H+-ATPase iso o m PMA1 unde con ol o he PMA1 p omo e . T ans o med yeas cells we e p e-cul u ed in 100 mL s e ile SGAH medium (7.04 g/L yeas ni ogen base, 19.8 g/L galac ose, 64 mg/L adenine, 64 mg/L his idine) o 3 days a 25°C and 150 pm. The p e-cul u e was ans e ed o 500 mL s e ile SGAH medium and u he incuba ed o 3 o 4 days. 100 mL om he cell cul u e was ans e ed o 1 L YPAD medium (10 g/L yeas ex ac , 20 g/L bac o-pep one, 20 g/L glucose, 20 mg/L adenine) and incuba ed a 25°C o 18-20 hou s. Recombinan yeas was ha es ed by 2-3 min o cen i uga ion a 3.000 x g and 4°C, ollowed by 2 imes wash in milli-Q wa e . Ha es ed cells we e incuba ed in 10% glucose o 10 min, on a shaking able, and cen i uged a 3.000 x g and 4°C. Cells we e e- suspended in homogenisa ion bu e (50 g/L glucose, 28.3% glyce ol, 0.1 M T is-HCl pH 7.25, 10 mM EDTA pH 8.0, 50 mM KCl, 1mM DTT, 200 µM PMSF, 2 µg/ml Peps a in A), and dis up ed wi h 165 g glass beads (500 µm) by uns in a BeadBea e (Biospec). The dis up ed cells we e cen i uged a 4°C o 5 and 15 min a 1.400 x g and 12.000 x g, espec i ely. The supe na an was collec ed and cen i uged a 251.000 x g o 1 h wi h 112 µM phenylme hylsul onyl luo ide (PMSF) and 1.1 µg/mL Peps a in A. The esul ing pelle was e-suspended in GTEK20 bu e (20% glyce ol, 10 mM T is-HCl pH 7.25, 25 mM KCl, 0.5 mM EDTA pH 8.0, 1 mM DTT, 0.2 mM PMSF, 2 µg/ml Peps a in A) and cen i uged o 45 min a 251.000 x g and 4°C. Pelle was hen e-suspended in STKED20 bu e (200 g/L suc ose, 40 g/L glucose, 50 mM T is-HCl pH 7.25, 50mM KCl, 1 mM EDTA pH 8.0, 1 mM DTT, 0.2 mM PMSF, 2 µg/ml Peps a in A), homogenised and dilu ed wi h STKED20 bu e . The plasma memb anes we e eco e ed a he in e ace o a 43%/53% (w /w ) s ep suc ose 32 g adien con aining suc ose in 50 mM T is-HCl pH 7.25, 50 mM KCl, 1 mM EDTA, 1 mM DTT. Cen i uga ion was done o 16 h a 154.000 x g and 4°C. The plasma memb ane ac ion was collec ed and dilu ed wi h GTEK20 bu e and cen i uged o 1 hou a 274.000 x g. Pelle was collec ed and homogenised in GTEK20 bu e and s o ed a -80°C. Sa co/Endoplasmic e iculum (SR) Ca2+-ATPase was p o ided in SR memb anes pu i ied by ex ac ion wi h a low concen a ion o deoxychola e (DOC) as desc ibed abo e. The pig kidney Na+/K+-ATPase pu i ica ion included a mild SDS ea men o isola ed mic osomes ollowed by a washing s ep and was kindly pe o med by Na alya Fedoso a, Aa hus Uni e si y and p epa ed as desc ibed in (131). In b ie , pieces o ou e medulla we e ex ac ed and cu in pieces and u he suspended and homogenized in ISE-bu e (25 mM imidazole, 250 mM suc ose, 1 mM EDTA pH 7.4). Mic osomes we e isola ed by di e en ial cen i uga ions. The inal pelle was suspended and homogenized in ISE-bu e and s o ed a -20°C. ATP Hyd olysis Inhibi ion ATPase ac i i y was de e mined by measu ing he amoun o libe a ed phospha e om ATP hyd olysis. The ATPase assay was pe o med in 96 well pla es in a inal eac ion olume o 60 µL. 0.1-0.2 µg/well o DOC ex ac ed SERCA memb ane o he Na+/K+-ATPase was used, while 1-2.5 µg/well was used o he Pma1 memb ane p epa a ion. Reac ions including p o ein memb ane p epa a ion and exogenously added compounds in a ½ log dilu ion concen a ion ange om 333 µM o 166 µM o 0.005 µM. We conduc ed he enzyma ic eac ions in he ollowing bu e s; Pma1 bu e : 17.5 mM MOPS-NaOH pH 7,7 mM MgSO4, 44 mM KNO3 ( acuola ATPase inhibi o ), 22 mM NaN3 (mi ochond ial ATPase inhibi o ), 0.22 mM Na2MoO4 (acid phospha ase inhibi o ); SERCA bu e : 9 mM MOPS-NaOH pH 7, 33 2.7 mM MgCl2, 0.1 mM CaCl2 and 72 mM KCl. Na+/K+-ATPase bu e : 30 mM MOPS-NaOH pH 7, 40 mM NaCl, 4 mM MgCl2 and 20 mM KCl. Reac ions we e s a ed by he addi ion o Na-ATP o a inal concen a ion o 2.5 mM (Pma1 and Na+/K+-ATPase) o 5 mM (SERCA), ollowed by 30 min incuba ion a 30°C. The amoun o libe a ed phospha e was de e mined calo ime ically a e addi ion o STOP solu ion (mix u e o L-asco bic acid, ammonium hep amolybda e e ahyd a e, and HCl o gi e inal concen a ions o 65 mM, 2.2 mM, and 189 mM, espec i ely) wi h 5 min incuba ion a RT ollowed by addi ion o a seni e solu ion (mix u e o NaAsO2, sodium ci a e dihyd a e, and ace ic acid o gi e inal concen a ions o 3.1 mM, 28 mM, and 141 mM, espec i ely). We measu ed abso p ion a 860 nm a e addi ional 30 min incuba ion a RT. Cell Viabili y, Apop osis and DNA Con en Assays ATP-based cell iabili y was de e mined using he CellTi e -Glo iabili y assay (P omega Co po a ion, Madison, WI, USA #G7573) and luminescence was measu ed using a Vic o X4 (Pe kin Elme , Wal ham, MA, USA). Apop o ic a e was quan i ied by s aining cells wi h Annexin V and p opidium iodide using a low-cy ome y comme cial ki (eBioscience™ Annexin V Apop osis De ec ion Ki APC, Wal ham MA, USA, # 88-8007-74). Cells we e analyzed by low cy ome y wi h a FACScan low cy ome e (Beckman Cul u e-Cy omics FC 500, Li e Sciences Di ision, Indianapolis, USA) and FlowJo V10 (T ee S a LLC, Ashland, OR, USA) analy ical so wa e. Cellula DNA con en was assessed by s aining wi h p opidium iodide (50 g/mL) and analyzed by low cy ome y. A leas 20,000 e en s we e acqui ed and all de e mina ions we e eplica ed a leas wice. 34 Cell Compe i ion Assay SKW-3/KE-37-GFP and MOLT16 we e co-cul u ed a 1:1 a io in RPMI 1640, 10% FBS, 1% P/S medium. 1 x 106 cells pe condi ion we e ea ed wi h CAD204520 a he ollowing concen a ions 2.5 and 5 µM and DMSO a 0.005% and 0.01% espec i ely and incuba ed a 37°C. A e 72 hou s, T-ALL cells we e washed in PBS, and s ained wi h a LIVE/DEAD Fixable Fa Red Dead Cell S ain (In i ogen, Li e Technologies, Ca lsbad, CA, USA, #L34973) o 30 min. Fluo escen signal was assessed by low-cy ome y [Beckman Cul u e- Cy omics FC 500 (Li e Sciences Di ision, Indianapolis, USA) and FlowJo V10 (T ee S a LLC, Ashland, OR, USA) analy ical so wa e]. A minimum o 20,000 e en s was collec ed o each biological sample. Expe imen s a e ep esen a i e o wo independen expe imen s. Compound Sou ces We ob ained he compounds o his s udy om he ollowing sou ces: DAPT (N-[N-(3,5- di luo ophenace yl)-1-alanyl]-(S)-phenylglycine) (Selleckchem, Hous on, TX USA, #S2215), hapsiga gin (Enzo Biochem, Inc., USA #BML-PE180-0005), dexame hasone, clobe asol p opiona e, lu icasone p opiona e and RU486 we e pu chased om MedChemExp ess EU (MCE) (MedChemT onica, Sweden, #HY-14648; #HY-13600; #HY-B0154; #HY-13683). Compound T ea men o Cell Lines and P ima y Cells Cells we e seeded in 384-well pla es (Co ning Li e Sciences Plas ic, Bed o d MA, USA, #3570) a he inal concen a ion o 0.02 x 106/mL pe condi ion. Small molecules we e added wi h a nanome ic dispense Tecan D300e (Tecan T ading AG, Swi ze land), and cellula iabili y was assessed a e 72 hou s o d ug ea men using a CellTi e -Glo ATP 35 assay (P omega Co po a ion, Madison, WI, USA, #G7573). IC50 and he a ea unde he cu e (AUC) we e calcula ed using G aphPad P ism so wa e (La Jolla, CA, USA). In acellula Calcium Measu emen Cy osolic Ca2+ concen a ion was measu ed using he Indo-1 AM p obe (The moFishe Scien i ic, Wal ham MA, USA, #I1223). Cells we e washed wice wi h a calcium ee solu ion (D-PBS Li e Technologies, Ca lsbad, CA, USA, #10010015) and loaded a 37°C in 5% CO2 o 30 min wi h 5 µM o Indo-1 AM. Then, cells we e washed wice wi h D-PBS and equilib a ed in RPMI 1640 (The mo Fishe Scien i ic, Wal ham MA, USA, Wal ham MA, USA #MT10040CV) wi h 10% FBS (Sigma-Ald ich, S . Louis, MO, USA, #F2442-500ML) and 1% penicillin-s ep omycin (The mo Fishe Scien i ic, Wal ham MA, USA, #3MT30002CI) o 5 min a 37°C. Baseline luo escence o Indo-1 AM loaded cells was acqui ed o 1 min LSR Fo essa X20 low cy ome e (BD Biosciences, San Jose, CA, USA). Subsequen ly DMSO (0.1%), CAD204520 1 µM, o hapsiga gin 1 µM we e added and measu emen was esumed o a o al o 10 min. Da a analysis was pe o med using FlowJo V10 (T ee S a LLC, Ashland, OR, USA) analy ical so wa e. ER Ca2+ elease and e-up ake was measu ed wi h he IonOp ix sys em (IonOp ix, Mil on, MA, USA). Ca2+ signals we e de ec ed by epi luo escence a e loading T-ALL cells (ALL/SIL, DND41) wi h Fluo-3-AM (10 µM; In i ogen, Ca lsbad, CA, USA) in PBS (Gibco, The mo Fishe Scien i ic, Wal ham, MA, USA) o 20 min, a RT. A e emo ing he luo opho e, cells we e washed wi h PBS o 30 min and hen placed (1x106; 1ml olume) in a chambe moun ed on he s age o an in e ed mic oscope (Nikon-Eclipse TE2000-U, Nikon Ins umen s, Flo ence, I aly). The eco ding s a ed wi h measu emen o baseline luo escence. Then, 1 µM CAD204520, 1 µM hapsiga gin, o DMSO (0.1%) we e manually 36 added wi h a pipe e, and he eco ding was con inued o up o 15 min (du ing he i s 7 min: 5 seconds o eco ding ollowed by 5 seconds o es ; in he emaining 8 min: 5 seconds o eco ding ollowed by 30 seconds o es ). Exci a ion leng h was 480 nm, wi h emission collec ed a 535 nm. The ollowing pa ame e s we e e alua ed: (i) peak luo escence no malized o baseline luo escence ( / 0_peak), (ii) ime a 50% o luo escence signal decay, measu ed om he peak ime ( ime_50%- / 0), and no malized luo escence compu ed a 3, 5, and 10 min om he peak ime ( / 0_3min, / 0_5min, and / 0_10 min). Wes e n Blo P o ein lysa es o wes e n blo ing we e incuba ed wi h an ibodies speci ic o g-sec e ase- clea ed NOTCH1 (Val1744, #4147 o #2421 Cell Signaling, Be e ly, MA, USA) o he C- e minus o NOTCH1 (#SC-6014 (C-20), San a C uz Bio echnology, San a C uz, CA, USA). Clea ed o m o Poly (ADP- ibose) polyme ase was de ec ed using an an ibody speci ic o he clea ed pep ide o PARP (#9541, Cell Signaling, Be e ly, MA, USA). The exp ession o SERCA iso o ms in ALL/SIL we e de ec ed using SERCA2 (#9580, Cell Signaling, Be e ly, MA, USA) and SERCA3 (#sc-81759, San a C uz Bio echnology, San a C uz, CA, USA) an ibodies while glucoco icoid ecep o exp ession was de ec ed using Glucoco icoid Recep o (D8H2) XP an ibody (#3660; Cell Signaling, Be e ly, MA, USA. Loading con ols we e pe o med wi h an ibodies speci ic o b-Ac in, (#BK3700S, Cell Signaling, Be e ly, MA, USA), GAPDH (#137179, San a C uz Bio echnology, San a C uz, CA, USA) o HSP90 (# sc-69703 (4F10)), San a C uz Bio echnology, San a C uz, CA, USA). E ec s on he endoplasmic e iculum s ess pa hway (ER s ess) we used he ollowing an ibodies: BiP, (#BK3177S), phospho-eIF2a (Se 51) (#9721S), eIF2a (#9722S) (Cell Signaling, Be e ly, MA, USA). Blo s we e de eloped using species speci ic luo escen an ibodies ob ained om 37 LI-COR (Biosciences, Lincoln, NE, USA) such as IRDye 680LT Goa an i-Mouse IgG (#925- 68020); IRDye 800CW goa an i- abbi IgG (#925-32211); IRDye 680RD goa an i- abbi IgG (#925-68071). Cell su ace NOTCH1 was e alua ed by s aining non-pe meabilized cells wi h monoclonal an i-human NOTCH1 an ibody (#FAB5317P, R&D, Minneapolis, MN, USA). Indi ec Immuno luo escence Mic oscopy DND41, REC-1 and ALL/SILL cells we e esuspended in PBS, spo ed on immuno luo escence slides (The mo Fishe Scien i ic, Wal ham, MA) by a cy ospin cen i uge (CR2000, Small P ime Cen i uge, Cen u ion) ixed o 10 min in 4% pa a o maldehyde (#28908, The mo Fishe Scien i ic, Wal ham MA, USA), pe meabilized in 0.2% T i on X-100 o 5 min, and blocked in 5% bo ine se um albumin o 1 hou . Then, he cells we e incuba ed wi h p ima y an ibodies agains ull leng h NOTCH1 (#SC-6014 (C-20) San a C uz Bio echnology, San a C uz, CA, USA o #ab44986 (A6) Abcam, Camb idge, Uni ed Kingdom), GOLGA1 (#SAB1409131, Sigma-Ald ich, S . Louis, MO, USA), and ATF6 (#37149, Abcam, Camb idge, Uni ed Kingdom). Alexa Fluo 488 (#A11029, In i ogen, Ca lsbad, CA, USA) and Alexa Fluo 568 (#A11036, In i ogen, Ca lsbad, CA, USA) we e used as seconda y an ibodies and cells we e incuba ed 1 hou a RT p o ec ed by he ligh . Nuclei we e s ained wi h DAPI (#D9542, Sigma-Ald ich, S . Louis, MO, USA). Co e slips we e moun ed wi h P olong Gold An i ade eagen (#P36934, The mo Fishe Scien i ic, Wal ham MA, USA). Images we e cap u ed using a EVOS FL mic oscope (The mo Fishe Scien i ic, Wal ham MA, USA) and analyzed using ImageJ so wa e (h p:// sbweb.nih.go /ij/). 38 Real- ime RT-PCR P ime s and p obes o eal- ime RT-PCR we e ob ained om Applied Biosys ems (Fos e Ci y, CA, USA) (RPL13A #Hs01926559_g1, MYC #Hs00153401_m1, DTX1 #Hs00269995_m1). P ime s and p obes used o glucoco icoid ecep o quan i a i e RT- PCR we e ob ained om The mo Fishe Scien i ic (Wal ham MA, USA) as ollowing. Fo human GR alpha, o wa d p ime : GAG-GAA-GTT-ATC-CTCTGC-CTC; e e se p ime : TGT-AAG-CAC-CAC-CTTCCT-GTC-T; p obe: 6FAM-TTC-CAA-CAG-TGA-GTCTGT-CAG- CGC-A-QSY; o human GR be a, o wa d p ime : GCT-GGA-TAA-TTA-GCA-TGG-GAT-G; e e se p ime : AAT-TGC-TCC-CTG-CCT-CTG-A; p obe: 6FAM-ATGAAG-GAA-AGC- CAC-GCT-CCC-T-QSY. The da a we e analyzed using he DDCT me hod and plo ed as pe cen age o ansc ip compa ed o ehicle. Values we e conside ed s a is ically signi ican a P < 0.05. Whole Exome Sequencing DNA was ex ac ed om abou 10 x 106 ALL/SIL o ALL/SIL hapsiga gin esis an using a P omega Maxwell™ ki as pe he manu ac u e ’s p o ocol (P omega Co po a ion, Madison WI, USA, #AS1010). A o al amoun o 1.0 µg genomic DNA pe sample was used as inpu ma e ial o he DNA lib a y p epa a ion. Sequencing lib a ies we e gene a ed using Agilen Su eSelec Human all exon ki (Agilen Technologies, CA, USA) ollowing manu ac u e ’s ecommenda ions and index codes we e added o each sample. B ie ly, agmen a ion was ca ied ou by hyd odynamic shea ing sys em (Co a is, Massachuse s, USA) o gene a e 180-280bp agmen s. Remaining o e hangs we e con e ed in o blun ends ia exonuclease/polyme ase ac i i ies and enzymes we e emo ed. A e adenyla ion o 3’ ends o DNA agmen s, adap e oligonucleo ides we e liga ed. DNA agmen s wi h liga ed 39 adap e molecules on bo h ends we e selec i ely en iched in a PCR eac ion. A e PCR eac ion, lib a y hyb idizes wi h liquid phase wi h bio in labeled p obe, a e which s ep omycin-coa ed magne ic beads a e used o cap u e he exons o genes. Cap u ed lib a ies we e en iched in a PCR eac ion o add index ags o p epa e o hyb idiza ion. P oduc s we e pu i ied using AMPu e XP sys em (Beckman Coul e , Be e ly, USA) and quan i ied using he Agilen high sensi i i y DNA assay on he Agilen Bioanalyze 2100 sys em and sequenced wi h Hiseq PE 150 (Illuminaâ, San Diego, CA, USA). Pai ed-end clean eads we e aligned o he e e ence genome (hg38) wi h Bu ows-Wheele aligne (B.W.A.). SAM ool was used o so and index he o iginal BAM iles and Pica d ma ked duplica es eads. Co e age and dep h we e calcula ed based on he inal BAM iles. I a ead o eads pai we e mapped o mul iple posi ions, B.W.A. will choose he mos likely posi ion. I wo o mo e likely posi ion we e p esen B.W.A. will choose one andomly. This mul iple hi s a egy has signi ican impac on SNP, INDEL and CNV de ec ion, and a ian calling accu acy. Following genomic a ian de ec ion, we pe o med anno a ion o a ian s wi h he ool ANNOVAR (132) in mul iple aspec s, including p o ein coding changes, a ec ed genomic egions, allele equency e c. Vi us P oduc ion and T ansduc ion o T-ALL Cell Lines 3 x 106 293T we e pla ed in 10 cm pla es and main ained in DMEM media (Li e Technologies, Ca lsbad, CA, USA, #11965118), 10% FBS (Sigma-Ald ich, S . Louis, MO, USA, #F2442-500ML), 1% penicillin-s ep omycin (The mo Fishe Scien i ic, Wal ham MA, #3MT30002CI) and incuba ed a 37°C wi h 5% CO2, un il sub con luen . Cells we e ans ec ed wi h 2 µg o pCMV-VSV-G en elope ec o , Del a 8.9 packaging plasmid and pXPR-011-GFP, a ec o exp essing a g een luo escen p o ein (GFP), acco ding o he 46 eplacemen wi h comme cially a ailable al e na i e he e oa oma ic and he e ocyclic sys ems, as well as py idines subs i u ed wi h small unc ional g oups capable o picking up pola in e ac ions. As a esul , he pipe idine analog 2-(2-pipe idyl)-6-( i luo ome hoxy)-1H- indole (Figu e 4BII) was iden i ied as he minimum pha macopho e wi h imp o ed SERCA ATPase po ency, selec i i y agains Na+/K+-ATPase, and a easonable ligand e iciency o 0.28. Op ions o di e si y in subs i u ion o indole C4-C7 we e limi ed and C6-OCF3 was ‘‘locked’’ o con inue explo a ion o mo e p omising poin s o subs i u ion. Fu he mo e, indole N1 had been ex ensi ely explo ed in a closely ela ed chemical p og am wi h an o e lapping pha macopho e, and o easons o conce n abou a ge selec i i y i was decided no o explo e he indole N1 chemical space wi h he iden i ied compound II. Con e sely, compound II was explo ed in R1 o he indole sys em and R2 o he pipe idine sys em (Figu e S1B) o easons de ailed below. Compounds wi h subs i u ion on pipe idine N1 (R2) wi h R1 = H we e subsequen ly p oduced and, among hem, CAD204522, CAD307496, and CAD204521 (Figu e 4B) showed a ious deg ees o Ca2+-ATPase ac i i y (Table S1). In e es ingly, CAD204521 (Figu e 4BIII; Table S1) was closely ela ed o a p e iously epo ed po en ungal H+-ATPase inhibi o , Compound 7 (144). Simila ly o Compound 7, CAD204521 showed an imp o ed po ency agains Ca2+ ATPase bu no a desi ed selec i i y o d ugabili y p o ile. Because indole C3 had he po en ial o p o ide Ca2+-ATPase selec i i y, we decided o explo e indole R1 wi h R2 = p-b omobenzyl (de i a i es o CAD204521, Figu e 4BIV). Fo example, CAD204519 was by a he mos po en inhibi o o Ca2+-ATPase; howe e , wi h an un a o able Na+/K+ Ca2+ selec i i y. Ne e heless, subs i u ions wi h ce ain hyd ophilic g oups on he R2 on pipe idine N1 (Figu e 4B; Table S1, CAD306750, CAD306749, and CAD204520) inc eased he selec i i y owa d mammalian SERCA. 47 No ably, CAD204520 (Figu e 4BV and S1C) p e e en ially inhibi ed he Ca2+-ATPase by educing i s ATP hyd olysis ac i i y wi h an IC50 o 0.34 ± 0.03 µM as compa ed wi h Na+/K+- ATPase (IC50 = 8.30 ± 0.94 µM) and H+-ATPase (IC50 = 26.90 ± 2.98 µM) (Figu e S1D; Table S1). Fu he mo e, CAD204520 displayed he o e all mos p omising d ug p ope ies o he syn hesized compounds wi h a calcula ed LogP o 4.4 and LogD7.4 o 2.2 (ACD/Labs 18.1.1). This compound was hus selec ed o u he s udies as a selec i e SERCA inhibi o . To assess he binding mode o CAD204520 o SERCA, we hen c ys allized i in complex wi h SERCA and de e mined he c ys al s uc u e a 3.4 Å esolu ion. The c ys als we e o he same space g oup as p e iously epo ed hapsiga gin-bound SERCA (PDB: 2AGV), and he o e all con o ma ion o SERCA bound o CAD204520 is e y simila o he hapsiga gin-bound o m. The CAD204520 ligand binds o a g oo e a he memb ane in e ace o SERCA, be ween ansmemb ane helices M1, M2, M3, and M4 (Figu es 4C and 4D), wi h wo pola in e ac ions o Asp59 on M1 (2.9 Å) and Asn101 on M2 (2.7 Å), and wi h se e al hyd ophobic in e ac ions in ol ing Leu61, Val62, Ile307, P o308, and P o312 (Figu e 4C). In e es ingly, he CAD204520 binding g oo e is di e en om ha o hapsiga gin (Figu e S1E), bu simila o he binding o o he SERCA inhibi o s, such as CPA (6) (Figu e S1F) and 2,5-di- -bu yl-1,4-benzohyd oquinone (DBHQ) (28) (Figu e S1G), and o ha o he Compound 7 p e iously epo ed by Bubli z e al. (144) (Figu e S1H). In ac , he indole sys em (co e s uc u e) o CAD204520 supe poses e y closely on he e ahyd oca bazole co e o Compound 7, including he cen al in e ac ion o he indole N1 ni ogen wi h Asp59. The mo pholinoe hyl g oup, in e ac ing wi h Asn101, occupies he same space as one o he wo al e na i e posi ions ound o he b omophenyl moie y o Compound 7 (Figu e S1H). In con as o Compound 7, howe e , he e is no in e ac ion wi h Asp245. Despi e his simila i y, CAD204520 does no induce he same o e all SERCA con o ma ion as Compound 7, bu a 48 con o ma ion almos iden ical o hapsiga gin-inhibi ed SERCA. The hapsiga gin-binding si e lies adjacen o he CAD204520 si e, sepa a ed by M3 (Figu e S1I). Collec i ely, hese da a show ha CAD204520 selec i ely binds SERCA in he same binding pocke as DBHQ, CPA, and Compound 7. This pocke has been iden i ied as he pa hway o Ca2+ ion en y in o he pump om he cy osolic side o he memb ane (29), and compound binding a his si e locks SERCA in a Ca2+- ee (so-called E2) con o ma ion. CAD204520 Rescues T-ALL Cells om Thapsiga gin Resis ance SERCA can be inhibi ed by di e en small molecules, such as hapsiga gin, DBHQ, 1,3- dib omo-2,4,6- is (me hyl-iso hio-u onium) benzene, and CPA. These compounds ha e speci ic binding si es in he ATPase p o ein and hence di e en inhibi o y mechanisms (146). A i s ques ion is whe he CAD204520 binding o SERCA mimics hapsiga gin ATPase inhibi o y kine ics o , a he , he wo molecules ac independen ly as p edic ed by s uc u al da a. To es ou hypo hesis, we ook wo di e en app oaches. Fi s , we gene a ed a T-ALL cell line (ALL/SIL) esis an o hapsiga gin (ALL/SIL R) by selec ing cells g owing unde inc easing concen a ion o his molecule. A app oxima ely days 90, 120, and 150, T-ALL cells displayed 2-, 10-, and 27- old inc eased IC50 alues, espec i ely (Figu e S2A). To ule ou ha his d ug esis ance was media ed by al e ed exp ession o he a ge , we demons a ed ha nai e and esis an cell lines showed simila le els o SERCA2 and SERCA3 p o eins (Figu e S2B). To e alua e o hapsiga gin-induced gene mu a ions wi hin he ATP2A1–3 genes, we pe o med whole-exome sequencing and limi ed ou analysis o single-nucleo ide exonic missense a ia ion wi h a Ph ed-scaled quali y sco e >30 (s anda d e o = 1/1,000 = 0.1%; accu acy 99.9%) (Figu e S2C). P e ious wo k had demons a ed 49 ha mu a ions occu ing in he hi d s alk (M3) segmen o SERCA de e mine he sensi i i y o ATPase o hapsiga gin (26, 147, 148). In pa icula , mu a ions in he M3 segmen be ween Asp254 and Leu260 inc ease he hapsiga gin concen a ions equi ed o inhibi ing SERCA by mo e han h ee o de s o magni ude (148, 149) (Figu e S2D). In e es ingly, in ALL/SIL R cells we iden i ied, wi hin he Asp254-Leu260 ho spo , a missense single-nucleo ide polymo phism occu ing in ATP2A2 exon 8 (c.G770T) caused a glycine257/ aline mu a ion in he M3 helix (Figu es 5A and S1I highligh ed in ed). No mu a ions occu ed in ATP2A1 (Figu e 5A, op panel), while missense mu a ions in ATP2A3 we e p esen bo h in he nai e and esis an lines, indica ing a p e-exis ing mechanism o allelic a iance (Figu e 5A, bo om panel). Simila ly, no acqui ed mu a ions we e iden i ied in SEC24A, a gene in ol ed in ER-Golgi p o ein a icking and p e iously iden i ied as an essen ial media o o hapsiga gin-induced cell dea h in a genome-wide CRISPR/Cas9 sc een in HAP1 cance cells (150). Acco ding o ou c ys al s uc u e, Gly257 aces a hyd ophobic pa o CAD204520 a a dis ance ha could p obably accommoda e a aline esidue wi hou in e e ing wi h CAD204520 binding (Figu e S1I). The in oduc ion o he bulky aline side chain will, howe e , e y likely limi he eedom o mo emen o he neighbo ing esidue Phe256, which has o swing sideways o accommoda e hapsiga gin binding (Figu es S1E and S1I), hus p o iding a po en ial explana ion o he esis ance e ec . Nex , we ea ed ALL/SIL nai e and esis an cells a ALL/SIL IC50 (as shown in he ollowing sec ions) concen a ions and demons a ed ha G257àV escues ALL/SIL cells om hapsiga gin-induced cy o oxici y while i does no in e e e wi h CAD204520 e ec s (Figu e 5B). Acco dingly, because CAD204520 binds o SERCA in a pocke simila o ha o CPA bu dis inc o ha o hapsiga gin, we an icipa ed ha he combined inhibi ion migh esul in a syne gis ic e ec wi h hapsiga gin bu no wi h cyclopiazonic acid. To a oid he 50 limi a ions and biases associa ed wi h any one algo i hm used o s udy d ug-d ug in e ac ions, we used comp ehensi e app oaches, including he Loewe addi i i y model, he Chou and Talalay index, and he Bi a ia e Response o Addi i e In e ac ing Doses (BRAID) analysis. The Loewe addi i i y is a commonly used dose-e ec -based model o quan i y a ze o-in e ac i e s a e o he combina ion o wo d ugs (151). The Chou-Talalay me hod (152) o d ug combina ion is based on he median-e ec equa ion and p o ides a mechanism-independen me hod o quan i a i e de e mina ion, combina ion index (CI), o d ug in e ac ions. A CI anging om 0.9 o 1.1 is conside ed addi i e, a CI < 0.9 indica es syne gy, and a CI > 1.1 esis ance. Finally, we used a esponse su ace me hod, he BRAID model o combined ac ion (153, 154). A κ BRAID index > 0 shows syne gy be ween he compounds es ed. Unlike mos me hods ha educe combina ion analysis o a simple decision be ween syne gy, addi i i y, and an agonism, su ace models use non-linea op imiza ion o i a esponse su ace model o he e ec s o combined compounds. We es ed CAD204520 and hapsiga gin bo h indi idually and in combina ions a he indica ed concen a ions o a o al o 60 combina o ial poin s in T-ALL cell lines and in p ima y NOTCH1-mu a ed T-ALL samples. We ound ha simul aneous exposu e o CAD204520 and hapsiga gin o 72 h esul ed in a obus syne gis ic inhibi ion o cell iabili y in T-ALL cells. Loewe, CI, and BRAID models es ablished a syne gis ic e ec a low-dose combina ions and suppo he no ion ha CAD204520 binds a a si e wi hin SERCA ha is dis inc om he hapsiga gin-binding si es (Figu es 5C–5E). Consis en wi h ou hypo hesis, combined CAD204520 and CPA ea men did no demons a e he same deg ee o syne gis ic ac i i y (Figu e S2E). Collec i ely, hese da a indica e ha G257V mu a ion in he M3 helix o SERCA do no in e e e wi h CAD204520 ac i i y and ha a g ea e an i-leukemia e ec may be achie ed 51 by he simul aneous binding o CAD204520 and hapsiga gin o hei espec i e si es in SERCA. CAD204520 Supp esses Leukemia G ow h in NOTCH1-Mu a ed T-ALL and MCL We p e iously demons a ed ha SERCA inhibi o s dec ease T-ALL g ow h bo h in i o and in i o (75). To alida e CAD204520 as a po en ial modula o o No ch-dependen cance s we ini ially es ed he e ec o CAD204520 in a panel o T-ALL o MCL cell lines ha con ain ac i a ing mu a ions in he HD o NOTCH1 and/o dele ions in he deg ada ion domain (PEST) (Figu e S3A). NOTCH1-mu a ed T-ALL (Figu e 6A) (ALL/SIL, CTV-1, DND41, PF382, and RPMI-8402) o MCL cell lines supp essed by GSI (REC-1) (Figu e 6B) (91) we e mo e sensi i e o CAD204520 as measu ed by inhibi ion o cell iabili y compa ed wi h NOTCH1 wild- ype umo cells (Figu es S3B and S3C). Se en y- wo hou s o CAD204520 ea men igge ed concen a ion-dependen apop osis as de e mined by he inc ease o Annexin V/PI+ cells (Figu e 6C) and he clea age o PARP p o eins (Figu e 6D). An addi ional pheno ypic consequence o NOTCH1 inhibi ion wi h GSI is ha T-ALL cells unde go cell-cycle a es (91). As shown in Figu e 6E, CAD204520 induced a G0/G1 a es p e e en ially in NOTCH1-mu a ed umo s (Figu e S3D), and oge he wi h he da a desc ibed abo e i suppo s he no ion ha CAD204520 inhibi s lymphoid-de i ed cance cells ca ying clinically ele an NOTCH1 HD o PEST mu a ions. CAD204520 Supp esses No ch1 Signaling NOTCH ecep o s unde go se e al p ocessing e en s, including a i s clea age by a u in- like con e ase (S1) in he ans-Golgi ne wo k ha gene a es ull-leng h he e odime s (155, 156) eady o be con eyed o he plasma memb ane (157). The co ec olding o hese 52 he e odime s equi es Ca2+ ha , in physiological condi ion, is igh ly egula ed ac oss he ER s o age by SERCA (68). To suppo he hypo hesis ha CAD204520-media ed SERCA inhibi ion impai s mu an NOTCH1 ma u a ion, we e alua ed he exp ession o NOTCH1 ull-leng h and ansmemb ane po ions o CAD204520- ea ed cells by wes e n blo ing. Lysa es om T- ALL cell lines ea ed wi h 5 μM CAD204520 o 24 h we e immunoblo ed wi h an an ibody speci ic o he cy oplasmic po ion o NOTCH1 ha ecognizes bo h unp ocessed NOTCH1 (FL-N1) (270 kDa) and he u in-p ocessed ansmemb ane subuni (TM-N1) (110 kDa). CAD204520 educed he le els o he u in-p ocessed ansmemb ane NOTCH1 subuni , bu no he unp ocessed ull-leng h NOTCH1 p ecu so , in mul iple T-ALL cell lines (Figu e 7A). As expec ed om ou s uc u al da a, combined CAD204520 and hapsiga gin ea men esul ed in an enhanced educ ion in ICN1 and TM-NOTCH1 le els (Figu e S4A). In addi ion, we demons a ed ha ea men o T-ALL wi h CAD204520 esul ed in a concen a ion-dependen dec ease in NOTCH1 exp ession on he cell su ace by low cy ome y (Figu e 7B). As expec ed, we did no obse e his e ec wi h a known GSI No ch inhibi o N-[N-(3,5-di luo ophenace yl)-1-alanyl]-(S)-phenylglycine (DAPT). Consis en wi h ou hypo hesis ha CAD204520 a ec s NOTCH1 ma u a ion a he han exp ession, NOTCH1 only dec eases a he su ace o he cells upon CAD204520 ea men bu co- localizes a he ER-Golgi in e media e compa men as shown by immuno luo escence co- localiza ion s udies (Figu es 7C and S4B–S4E). An immedia e consequence o he dec emen in NOTCH1 on he su ace o he cells is he educ ion o he ca aly ic ac i i y o he g-sec e ase complex because o he lack o NOTCH1 subs a e. He e, we would expec a educ ion in he le el o ICN1. Indeed, CAD204520 ul ima ely leads o loss o ICN1 (Figu e 7D) and esul s in he supp ession o NOTCH1 a ge genes MYC and DTX1 as measu ed 53 by RT-PCR (Figu e 7E). Fu he mo e, es ing CAD204520 in MCL NOTCH1-mu a ed cells yielded esul s compa able wi h he one desc ibed in T-ALL, sugges ing a conse ed mechanism ac oss di e en NOTCH1-mu a ed cance s (Figu e S4D-F). In summa y, hese da a show ha CAD204520 inhibi s No ch1 ma u a ion, demons a ing ha SERCA inhibi o s wi h a binding mode di e en om hapsiga gin can e icien ly supp ess NOTCH1 ma u a ion. CAD204520 P e e en ially Inhibi s NOTCH1-Mu a ed Cance s SERCA inhibi o s inc ease he No ch he apeu ic index by a ge ing clinically ele an NOTCH1 mu a ions in leukemia cells (75, 119, 158). In ac , leukemia cells ca ying NOTCH1 alleles wi h HD mu a ions a e mo e sensi i e o SERCA inhibi ion han cells wi h wild- ype NOTCH1 alleles (75, 119). To e i y he hypo hesis ha CAD204520 p e e en ially a ge s mu an NOTCH1, we used wo T-ALL cell lines ca ying he same (8; 14) (q24; q32)/TRAD@-MYC ansloca ion bu di e en No ch mu a ional s a us (Figu es 8A and S5A). SKW-3/KE-37 ha bo s an isola ed NOTCH1 mu a ion in he PEST domain, while MOLT16 is NOTCH1 wild ype (91, 159). Fi s , we de e mined ha he mu an T-ALL cell line was mo e sensi i e o CAD204520 g ow h inhibi ion as measu ed by an ATP-based cell iabili y assay (Figu e S5B). To alida e his obse a ion, we es ablished a low cy ome y-based compe i ion assay. SKW-3/KE-37 we e ansduced wi h a GFP len i i al exp essing ec o and co-cul u ed wi h MOLT16 T-ALL cells in a 1:1 a io. Nex , we ea ed SKW-3/KE-37-GFP and MOLT16 cocul u ed cells wi h inc easing concen a ions o CAD204520 and demons a ed ha he mu a ed T-ALL cell line, SKW-3/KE-37-GFP, was mo e sensi i e o g ow h supp ession compa ed wi h wild- ype MOLT16, as quan i ied by low cy ome ic analysis o ali e e sus dead cells (Figu e 54 8B). Analysis o caspase-3 and -7 ac i i ies indica es ha he NOTCH1 mu a ional s a us sensi izes cells o CAD204520-media ed apop o ic cell dea h (Figu e 8C). Consis en wi h he hypo hesis ha SKW-3/KE-37 elies on No ch signaling o g ow h and su i al, we obse ed a dec emen o NOTCH1 p o ein only in mu an T-ALL cells compa ed wi h wild- ype cells (Figu e 8D). To u he suppo he p eclinical de elopmen o CAD204520, we es ed i (dose ange = 0.6–8 μM) in a collec ion o T cell lymphoblas s isola ed om T-ALL pa ien s. As shown in Figu e 8E, CAD204520 p e e en ially a ec s T-ALL iabili y compa ed wi h no mal lymphocy es. P ima y blas s, de i ed om a pa ien su e ing om a NOTCH1-mu a ed T- ALL, exposed o 5 μM CAD204520, apidly unde wen apop osis (Figu e S5C). In addi ion, T-ALL p ima y cases o which we con i med a NOTCH1 mu a ion (no. 1 NOTCH1 ex 27, c.5101G > C p.A1701P; no. 2 NOTCH1 ex 26, c.4793G > C p.1598P and FBXW7 ex 9, c.1514G > T p.R505L) we e mo e sensi i e o CAD204520 compa ed wi h NOTCH1 wild- ype B cell ALL (Figu e 8F). Collec i ely, hese esul s indica e ha CAD204520 e ains an i- umo ac i i y p e e en ially in cells ca ying NOTCH1 alleles wi h HD o PEST mu a ions, holding g ea p omise o CAD204520 u u e de elopmen agains his indica ion. Consequences o Ca2+ Release upon CAD204520 T ea men A consequence o SERCA inhibi ion is he ise o in acellula Ca2+ ollowed by he deple ion o Ca2+ s o ed in he ER. ER Ca2+ exhaus ion igge s a numbe o seconda y e en s, including he ac i a ion o he un olded p o ein esponse (UPR) pa hway (160), he ac i a ion o s o e-ope a ed Ca2+ en y (161), and ul ima ely cell dea h (162). 55 To quan i y he consequences o CAD204520 o hapsiga gin ea men a he le el o cy osolic Ca2+, we ans e ed ALL/SIL o DND41 in Ca2+- ee media and loaded wi h Indo- 1 a a iome ic sensi i e indica o luo escen dye o measu ing in acellula Ca2+. As shown in Figu e 9A compa ed wi h DMSO, CAD204520 sligh ly inc eases cy osolic Ca2+. Howe e , i compa ed wi h he hapsiga gin e ec , he ex en o he inc ease appea s modes wi h b oad and la peaks. In ac , hapsiga gin causes a sha p ise in Ca2+ concen a ion a ~ 200 s upon d ug injec ions. The nex ques ion is whe he SERCA is s ill able o e-load Ca2+ om he cy osol inside he ER upon CAD204520 ea men . This hypo hesis would explain why he inc ease o Ca2+ upon CAD204520 ea men is mode a e and why hapsiga gin igge s delayed on- a ge Ca2+ e ec s such as UPR ac i a ion and apop osis (163). In his case we used a di e en app oach and measu ed Ca2+ Fluo-3 AM epi luo escence using an IonOp ix sys em. This app oach is ideal o measu e luc ua ions o ER Ca2+. As epo ed in Figu e 9B, he peak luo escence ( / 0_peak) was simila in he h ee g oups o cells, indica ing ha he di e en compounds did no modi y he Ca2+ elease om he ER. Con e sely, he luo escence signal decay was signi ican ly p olonged in hapsiga gin- ea ed cells in compa ison wi h bo h CAD204520 and DMSO ( ime_50%- / 0; p < 0.05). In acco dance wi h his inding, he luo escence compu ed a 3, 5, and 10 min om he peak ime ( / 0_3 min, 5 min, and 10 min) o he a ea unde he dose cu e calcula ed wi hin he same ime ame (con ol e sus CAD204520 Δmean = 0.4188; con ol e sus hapsiga gin Δmean = -2.658; CAD204520 e sus hapsiga gin Δmean = -3.077), was signi ican ly highe only in he hapsiga gin g oup (p < 0.05 and p < 0.0001, espec i ely), sugges ing ha CAD204520 did no delay he cy osolic Ca2+ eup ake. As p e iously men ioned, in addi ion o he e ec s on Ca2+ dynamics, hapsiga gin and hapsiga gin analogs ac i a e he ER- ela ed s ess pa hway o he UPR (164). To compa e 62 DISCUSSION Al hough he p ognosis o T-ALL has imp o ed o e he las wo decades, he ou come o T-ALL pa ien s wi h p ima y esis an o elapsed disease emains poo (173, 174). The e o e, cu en esea ch goals a e ocused on he iden i ica ion o a ge s o de elop mo e e ec i e and less- oxic an i-leukemic agen s (100, 175-177). Se e al s udies s ongly suppo he de elopmen o No ch inhibi o s o a ge ed he apy in hema ological malignancies and solid umo s whe e No ch signaling is de egula ed (178). Fo example, pan No ch pa hway an agonism wi h GSIs educes leukemia g ow h in mu an cance cell lines and in mouse models (91, 179). Thus, modula o s o No ch would be expec ed o ha e clinical e icacy pa icula ly in T-ALL whe e ecu en NOTCH1 mu a ions a e common and cance dependency has been well es ablished. Howe e , p olonged supp ession o he canonical No ch pa hway in no mal issue may cause dose-limi ing gas oin es inal oxici y (103) o inc ease he isk o skin cance s (180, 181), unde sco ing he need o new he apeu ic modali ies o p e e en ially supp ess he oncogenic signal. In ecen yea s, we ha e pu sued his app oach and demons a ed ha selec i e inhibi o s o SERCA, such as hapsiga gin and CPA, uniquely among No ch inhibi o s, p e e en ially a ec mu a ed NOTCH1 p o eins compa ed wi h he wild- ype ones (75, 119). Thapsiga gin, a plan -de i ed sesqui e pene-g-lac one, has been used ex ensi ely as a pha macological ool o igge Ca2+-dependen and UPR pa hways in se e al disease models (182). Because he inc ease o cy osolic Ca2+ and sus ained UPR ac i a ion (ER s ess) a e impo an media o s o apop osis, SERCA inhibi o s ha e been conside ed o cance he apies (183). Howe e , la ge-scale isola ion om Thapsia o scalable syn hesis o hapsiga gin is complex, equi ing a 5- o 42-s ep p ocess depending on he p o ocol used 63 (184-186). Simila ly, he syn hesis o hapsiga gin-based de i a i es p esen s signi ican challenges. In ac , hapsiga gin i sel possesses a polyoxygena ed 5-7-5 icyclic co e linked o ou di e se es e g oups and eigh s e eogenic cen e s no sui able o s uc u al modeling (184, 187). S uc u e-ac i i y s udies e ealed ha only ew hapsiga gin g oups, o example, he es e bond a O(8), can be hyd olyzed o gene a e in e media e de i a i es ha can be used o conjuga ion wi h a pep ide (188, 189) o wi h a clea able es e linkage (119). In addi ion, modi ica ion o he hapsiga gin es e acyl g oup, o o he lac one ca bonyl, signi ican ly educes hapsiga gin ac i i y in cells o biochemical assays, p e en ing hei b oad applicabili y in cance (190, 191). A u he limi a ion is ha na i e hapsiga gin is no ac able as a he apeu ic agen due o expec ed Ca2+ shi s ha can be p e en ed, o example, by c ea ing inac i e p o-d ugs ac i a ed in a hys o-speci ic manne (116, 192). This is he mode o ac ion o mipsaga gin, a hapsiga gin de i a i e cu en ly unde going clinical ials o solid umo s (183). Ou g oup has de eloped JQ-FT, a ola e- hapsiga gin de i a i e ha le e ages he dependency o leukemia cells on ola e me abolism o di ec he inhibi o in o T-ALL cells (119). Ano he s a egy is o exploi analogs ha possess an enhanced selec i i y owa d SERCA iso o ms p e e en ially exp essed in cance cells (115, 193), while keeping he ac i i y o SERCA2a, he majo ca diac iso o m, una ec ed (194-196) (Figu e 3). An al e na i e is he de elopmen o small molecules ha e ain SERCA inhibi o y capaci ies bu ha e only ansien e ec s on cy osolic Ca2+ shi s. This idea eme ged om ecen s udies om he labo a o y o Mølle and colleagues ha challenged he consensus idea ha he ele a ion o cy osolic Ca2+ — a he han he deple ion o ER Ca2+ — led o he cell dea h induced by hapsiga gin and analogs (164, 197). Con a y o wha is gene ally hough , he apid ise o cy osolic Ca2+, as obse ed wi h hapsiga gin, and i s ole in he sho - e m 64 side e ec on ca diac con ac ili y, is no equi ed o apop osis a e SERCA inhibi ion. I is a he he ER Ca2+ deple ion and sus ained UPR ac i a ion ha con ibu es o cell dea h (164). The e ec o a gi en SERCA inhibi o on cy osolic and ER Ca2+ le els depends s ongly on i s molecula mechanism o in e ac ion wi h he ATPase. Fo example, he hapsiga gin de i a i e subs i u ed wi h a 12-aminododecanoyl linke , Boc-8ADT, did no show measu able changes in Ca2+ le els e en hough i s ongly inhibi ed SERCA ATPase ac i i y (164) leading o apop osis in LNCaP cells (198). This is p obably due o he e y slow binding kine ics o his compound leading o a slow ne leakage o Ca2+ om he ER, which likely enables he main enance o cons an , s able cy osolic Ca2+ le els. O he possible causes o he lack o cy osolic Ca2+ peaks a e a mode a e dec ease in SERCA’s Ca2+ a ini y o a esidual Ca2+ anspo ac i i y in he p esence o he compound. I is emp ing o specula e ha o he SERCA inhibi o s ha ha e ad anced o clinical es ing migh ha e a simila mode o ac ion. Cu cumin, a small molecule de i ed om he u me ic spice ha s abilizes SERCA in he E1 con o ma ional s a e has been ex ensi ely es ed in mul iple cance models and clinical ials (199) wi hou causing majo ca diac e en s. Cispla in is a widely used pla inum- con aining compound ha , among o he e ec s, inhibi s SERCA and Na+/K+-ATPase simul aneously (200). Gi en he la ge numbe o o a ing bonds in CAD204520, slow binding kine ics o SERCA, as wi h Boc-8ADT, can also be an icipa ed. F om ou s uc u al da a, he in e ac ion o CAD204520 wi h SERCA in ol es only wo pola con ac s, and one single hyd ophobic con ac wi hin a dis ance o 3 Å. O e all, he in e ac ion looks su p isingly ‘‘loose’’, pe haps indica ing a concen a ion-dependen compe i ion wi h Ca2+ binding and anspo a he han an i e e sible inhibi ion. 65 Ou da a also show ha CAD204520 binds o SERCA di e en ly om hapsiga gin: i occupies a pocke be ween he ansmemb ane helices M1, M2, M3, and M4 o SERCA, whe eas hapsiga gin binds be ween M3, M5, and M7 (59). This obse a ion ag ees wi h ou inding ha hapsiga gin, bu no CPA, co- ea men enhances CAD204520’s inhibi o y e ec , a ea u e ha can be used o u he medicinal chemis y op imiza ion. In his ega d, howe e , CAD204520 main ains he same hapsiga gin ‘‘p ope y’’ o p e e en ially al e mu a ed NOTCH1 a icking. Rema kably, his abili y has no ye been explo ed in wo o he mos ecen ly syn hesized pu a i e SERCA inhibi o s: he na u al icyclic cle odane di e pene casea in J (201) o e hyl 2-amino-6-(3,5-dime hoxyphenyl)-4-(2-e hoxy-2- oxoe hyl)-4H-ch omene-3-ca boxyla e (CXL017) (202), bo h ac i e in T-ALL cell lines. The nex ques ion is whe he CAD204520 ac i i y has limi a ions in i o due o Ca2+ shi s. Fo example, mice exposed o a hapsiga gin analog, L12-ADT, a 0.8 mg/kg, die wi hin 8 h, pu a i ely om ca diac oxici y (73). Ca diac SR Ca2+ ATPase (SERCA2a) plays a cen al ole in myoca dial con ac ili y. SERCA2a ac i ely anspo s Ca2+ in o he SR and egula es cy osolic Ca2+ concen a ion, SR Ca2+ load, and hus he a e o con ac ion and elaxa ion o he hea (203). The amoun o Ca2+ elease om he SR, dic a ing he ex en o cell sho ening, is also a s eep unc ion o SR Ca2+ con en (204). I ollows ha pha macological inhibi ion o SERCA2a ac i i y should educe he ampli ude o he ansien calcium and he a e o SERCA-media ed Ca2+ emo al, esul ing in al e ed ca diomyocy e mechanics, as we obse ed in isola ed unloaded en icula myocy es exposed o CAD204520 o hapsiga gin. Howe e , he impai men o cellula con ac ile pe o mance and Ca2+ dynamics was mo e p onounced a e hapsiga gin incuba ion compa ed wi h CAD204520 exposu e (80%–90% educ ion in unc ional pe o mance e sus 25%–30%, on a e age), 66 indica ing ha CAD204520 should ha e a be e he apeu ic window han hapsiga gin in i o. An impo an ques ion is whe he and o wha ex en he dep essed ca diomyocy e unc ion seconda y o pha macological inhibi ion o SERCA2 ac i i y would ansla e in o dec eased ca diac unc ion in i o. Based on p e ious expe ience om ou g oup in a a model o induced ca diomyopa hy (205), a 20%–30% decline in cellula mechanics ex i o esul s in a compa able mode a e hemodynamic impai men in he in ac animal. In ac , while CAD204520 exe s an an i-leukemia e ec in i o i does no induce hea ailu e in he wo di e en mouse models (BALB/c CD1 and IL2-NSG) used o his s udy. Al hough mos NOTCH1 mu a ions a e ound in exons 26 and 27 coding o he HD egion, mu a ions in he PEST domain a e p esen in 20%–30% o umo s esul ing in an inc eased No ch ac i a ion due o he p olonged s abiliza ion o ICN1 (91). Ac i a ing mu a ions clus e ed in he PEST sequence ha e been desc ibed in CLL and in MCL, and se e al e o s a e ongoing o a ge NOTCH1 in hese diseases (92, 118). Ou s udy demons a es ha CAD204520 is ac i e in cell lines ca ying a PEST mu a ion (SKW-3/KE-37). This esul suppo s es ing SERCA inhibi o s in disease models wi h his ecu en abno mali y, such as CLL (206) and MCL (92). To his end, we ex ended es ing CAD204520 in he REC-1 MCL cell line, one o he ew ep esen a i e models o NOTCH1-dependen MCL (92) ca ying a H2428P s*7 PEST mu a ion. We showed ha REC-1 is sensi i e o CAD204520 inhibi ion compa ed wi h NOTCH1 wild- ype MCL lines. In REC-1, CAD204520 educes No ch ac i a ion wi h a mechanism simila o he one obse ed in T-ALL. Because in MCL NOTCH1 mu a ions a e associa ed wi h signi ican ly sho e su i al a es (92, 207), he de elopmen o No ch- a ge ed he apy may ep esen an e ec i e s a egy o ackle his agg essi e disease. 67 Finally, since SERCA inhibi o s display a a o able he apeu ic index by a ge ing mu a ed NOTCH1 p o eins, he de elopmen o new SERCA inhibi o s, such as CAD204520 is a easonable s a egy o NOTCH1-mu a ed malignancies. Fo his eason, o an icipa e he po en ial mechanism o esis ance o SERCA inhibi o s can imp o e he deep cha ac e iza ion o his class o molecule and speed up he ansla ion in o a clinical se ing. To his pu pose, we pe o med a small molecule sc eening on wo T-ALL cell lines, espec i ely sensi i e and esis an o he e ec o hapsiga gin, and iden i ied glucoco icoids among he op classes wi h high ac i i y in he esis an cell line. The modula ion o SERCA ac i i y due o he ho spo mu a ion in he hapsiga gin binding si e induced an up egula ion o glucoco icoid ecep o wi h he subsequen e e sal o s e oid esis ance. Fu he mo e, glucoco icoids showed a syne gis ic ac i i y wi h SERCA inhibi o s especially in he esis an cell line, pa ing he way o a be e unde s anding o SERCA ac i i y and modula ion in NOTCH1-mu a ed cance . 68 CONCLUSIONS Modula ion o in acellula Ca2+ homeos asis plays c i ical oles in key p ocesses ha egula e cellula su i al, g ow h, di e en ia ion, me abolism, and dea h in no mal and cance cells. Thus, i is no su p ising ha se e al an i-cance agen s supp ess p o-su i al and ac i a e p o-apop o ic pa hways h ough modula ion o Ca2+ signaling-dependen mechanisms. This is, o example, he case o chemo he apeu ics such as cy o oxic alkyla ing agen s (208) o an i-me aboli es ha ely on a Ca2+ signaling componen o induce cance cell dea h (209). Simila ly, na u al compounds including alkaloids, la onoids, di e penoids, and polyphenolics ha e been ex ensi ely in es iga ed o hei abili y o modula e in acellula Ca2+ concen a ion and pa icipa e in apop o ic signaling pa hways. Among hem SL, such as hapsiga gin ha e been long ega ded as a ge compounds o d ug de elopmen . In ac , hapsiga gin has a b oad spec um o g ow h supp essing ac i i y in se e al umo ypes including poo ly di iding cells (210). Howe e , we ha e demons a ed he SERCA inhibi ion may e icien ly con ol he a icking o NOTCH1 and ha his blockade can be achie ed wi hou causing o e ca diac oxici ies in p eclinical leukemia models. Impo an ly he e ec s o SERCA supp ession can be escued by he o e exp ession o unp ocessed NOTCH1 pep ides such as ICN1 indica ing ha he an i-leukemia e ec is on a ge o No ch inhibi ion a he han o mo e gene ic Ca2+ luxes. An impo an s anding ques ion is why mu a ed NOTCH1 appea s mo e sensi i e o SERCA supp ession compa ed o wild ype iso o m o o he p o eins mo e b oadly. One hypo hesis o explain NOTCH1 and SERCA unc ional dependency is by mechanisms o co- egula ion. I has been p e iously shown ha p esenilin (PSEN) and SERCA co-localize in he ER (211). Since PSEN1 is a key egula o o NOTCH1 ma u a ion and p e e en ially binds FL-N1 polypep ides p ocessed h ough he ER, i is possible ha NOTCH1-PSEN1-SERCA a e 69 pa o a co- unc ional p o ein complex. In e es ingly, in a ecen pape , ea men o T-ALL cell lines wi h he selec i e PSEN1 inhibi o MRK-560 inhibi ed mu an NOTCH1 p ocessing and led o cell cycle a es . MRK-560 ea men dec eases leukemia bu den and inc eased o e all su i al wi h no associa ed gu oxici y in T-ALL pa ien -de i ed xenog a s in i o sugges ing ha , simila o SERCA inhibi ion, dis up ion o PSEN1 may p e e en ially a ec mu a ed p o eins. The second hypo hesis is a Ca2+ media ed one. In ac , Malecki and colleagues p e iously demons a ed ha clinically ele an ac i a ing NOTCH1 HD mu a ions des abilize he NOTCH nega i e egula o y egion and ha e dele e ious e ec s on NOTCH1 olding and ma u a ion. Because EGF and LNR epea s o NOTCH1 ely on Ca2+ o olding and ac i a ion, i may be possible ha changes in ER Ca2+ may p e e en ially impai uns able NOTCH mu an p o eins (212) compa ed o wild ype p o iding a he apeu ic window o SERCA inhibi o s. Finally, a hypo hesis no ye explo ed o explain FL-N1 accumula ion a concen a ions no su icien o igge he gene al mechanism o UPR is h ough a Ca2+ media ed ansc ip ional ac i a ion o inhibi o s o u in-like p o eases. This would explain o example why CAD204520 e icien ly a ge cance s wi h isola ed PEST dele ions ha would no be p edic ed o be uns able gi en a no mal LNR and HD p o ein sequence. In conclusion, his s udy p esen s CAD204520 as an o ally bioa ailable SERCA inhibi o wi h ole able o - a ge oxici y in NOTCH1-dependen umo s. 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Molecula pha maceu ics. 2013;10(11):4358-66. 94 [L1594PΔPEST] and RPMI-8402 [ins1584PVELMPPE]). The blo was incuba ed wi h an an ibody agains he C e minus o NOTCH1 ha ecognizes bo h he u in-p ocessed NOTCH1 ansmemb ane subuni (TM) and he unp ocessed NOTCH1 p ecu so (FL). B) E ec o 24 h o CAD204520 and GSI (N-[N-(3,5-di luo ophenace yl)-1-alanyl]-(S)- phenylglycine [DAPT]) ea men s on NOTCH1 cell su ace s aining as assessed by low cy ome y. C) E ec o CAD204520 and GSI (DAPT) ea men (24 h) on he subcellula localiza ion o NOTCH1. Immuno luo escence images o pe meabilized ALL/SIL incuba ed wi h an i- No ch1 (C20- ed) and an i-Golgin (g een) a e shown. Co-localiza ion is indica ed by yellow signal. Scale ba , 100 µm magni ica ion. D) Wes e n immunoblo showing he exp ession o clea ed NOTCH1 (ICN1) in ALL/SIL, DND41, and RPMI-8402 cells ea ed a he indica ed concen a ions o CAD204520 o 24 h. HSP90 was used as a loading con ol. E) CAD204520 ea men o 24 h down egula es exp ession o NOTCH1 a ge genes in ALL/SIL, DND41, and RPMI-8402 T-ALL cells as assessed by qRT-PCR. E o ba s indica e he mean ± SD o ou eplica es. Da a we e analyzed using he ΔΔCT me hod and plo ed as a pe cen age ela i e o he con ol gene RPL13A. S a is ical signi icance (***p ≤ 0.001, ****p ≤ 0.0001) was de e mined by one-way ANOVA using Bon e oni’s co ec ion o mul iple compa ison es ing. GSI (DAPT) was used as a posi i e con ol. Figu e 8. NOTCH1 Mu a ion Sensi izes T-ALL Cells o CAD204520 Inhibi ion 95 A) In e phase and me aphase FISH, wi h he LSI MYC p obe, show spli signals be ween de (8) ( ed signal) and de (14) (g een signal), in he MOLT-16 (le ) and SKW-3/KE-37 ( igh ) cell lines. (b) SKW-3/KE-37 has wo de (8). B) Le : cell-based compe i ion assay. SKW-3/KE-37 and MOLT16 we e ansduced wi h a GFP-con aining ec o o an emp y con ol ec o , espec i ely, and co-cul u ed a a 1:1 a io. Righ : no malized e ec o CAD204520 on cellula iabili y in co-cul u ed SKW-3/KE- 37-GFP and MOLT16 cells ea ed o 72 h. E o ba s deno e he mean ± SD o wo eplica es o ehicle- ea ed (DMSO) cells and o CAD204520- ea ed cells. S a is ical signi icance (*p ≤ 0.05) was de e mined by one-way ANOVA using Bon e oni’s co ec ion o mul iple compa ison es ing. C) Caspase-3/-7 luminescence old induc ion in SKW-3/KE-37 and MOLT16 cells. E o ba s deno e he mean ± SD o six eplica es o ehicle- ea ed (DMSO) cells and o CAD204520- ea ed cells. S a is ical signi icance (***p ≤ 0.001) was de e mined by one-way ANOVA using Bon e oni’s co ec ion o mul iple compa ison es ing. D) E ec o CAD204520 ea men o 24 h on NOTCH1 (N1) p ocessing and ac i a ion in SKW-3/KE-37 and MOLT16 cell lines. The immunoblo was s ained wi h an an ibody agains he C e minus o NOTCH1 ha ecognizes he u in-p ocessed NOTCH1 TM and he unp ocessed NOTCH1 p ecu so (FL). HSP90 was used as a loading con ol. E) E ec o he CAD204520 in p ima y T-ALL cells (n = 9) o isola ed lymphocy es (n = 6). The whiske plo ep esen s he e ec o small molecules on cellula iabili y calcula ed using he a ea unde he cu e (AUC) model o log- ans o med dose- esponses da a using G aphPad .7. The line in he whiske diag am ep esen s he AUC median. The uppe edge (hinge) indica es he 75 h pe cen ile o he da ase , and he lowe hinge he 25 h pe cen ile. 96 The ends o he e ical line show he minimum and he maximum da a alues. S a is ical signi icance (***p ≤ 0.001) was de e mined by a non-pa ame ic es (Mann-Whi ney). F) No malized e ec o he CAD204520 in p ima y NOTCH1-mu a ed T-ALL cells (n = 2) o p ima y B-ALL cells (n = 2) on cellula iabili y. E o ba s deno e he mean ± SD o ou eplica es. S a is ical signi icance compa ing each T-ALL e sus B-ALL case o each dose (***p ≤ 0.001, ****p ≤ 0.0001) was de e mined by a non-pa ame ic es (Mann-Whi ney). Figu e 9. E ec s o CAD204520 on Ca2+ and UPR Ac i a ion A) Indo-1 AM luo escence aces o T-ALL cells loaded wi h 5 μM o Indo-1 AM and ea ed wi h DMSO, CAD204520 1 μM, o hapsiga gin 1 μM. Baseline and pos - ea men luo escence is indica ed by a black a ow. Cells we e acqui ed o a minimum o 10 min on an LSR Fo essa X20 low cy ome e . B) Time cou se o ER calcium elease and eup ake aces eco ded in DMSO, CAD204520, and hapsiga gin T-ALL- ea ed cells. Each ace is ep esen a i e o i e (DMSO) o ou (CAD204520 and hapsiga gin) independen expe imen s. In g een he a ea unde he cu e (AUC). Values a e epo ed as mean ± SEM; / 0_peak, peak luo escence no malized o baseline luo escence; ime_50%- / 0, ime a 50% o luo escence signal decay measu ed om he peak ime; / 0_3min, 5min, and 10 min, luo escence compu ed a 3, 5, and 10 min om he peak ime. *p < 0.05 e sus DMSO; #p < 0.05 e sus 1 μM CAD204520. S a is ical signi icance was de e mined by a K uskal-Wallis es and di e ences among g oups we e de e mined by a Mann-Whi ney non-pa ame ic es . C) E ec o CAD204520 and hapsiga gin ea men o 24 h in ALL/SIL and DND41 cell lines. The blo was s ained wi h an an ibody agains he C e minus o NOTCH1 ha 97 ecognizes he u in-p ocessed NOTCH1 TM and he unp ocessed NOTCH1 p ecu so (FL), an an ibody ha ecognizes he clea ed NOTCH1 (ICN1), P-eIF2α, o al eIF2α, BiP, and HSP90 used as a loading con ol. D) E ec o CAD204520 and hapsiga gin ea men o 24 h on ATF6 in ALL/SIL cell line. Immuno luo escence o pe meabilized ALL/SIL cells s ained wi h ATF6 (g een) is shown. Cell nuclei we e s ained wi h DAPI (blue). Scale ba , 100 mm. E) E ec s o CAD204520 (le ) and hapsiga gin ( igh ) on cell iabili y a e 72 h o ea men s in HL-1 and ALL/SIL cell lines. E o ba s deno e ± SD o a minimum o wo eplica es. (F) E ec o CAD204520 and hapsiga gin ea men o 24 h in HL-1 cell lines. The blo was s ained wi h BiP. β-Ac in was used as a loading con ol. Figu e 10. E ec s o CAD204520 on P eclinical Model o T-ALL A–C) Le panels: e ec o CAD204520 ea men on a ca diomyocy e mechanics. Single expe imen s a e ep esen ed by wo do s in e connec ed by a solid line. Speci ically, he line be ween he do s connec s he quan i ica ion o maximal a e o sho ening (A) (-dl/d max), maximal a e o e-leng hening (B) (+dl/d max), and ac ion o sho ening (C) (FS%), be o e and a e he CAD204520 (5 μM) o hapsiga gin (200 nM) ea men compa ed wi h con ol (Con ol). (A–C) Righ panels: mean pe cen age e ec o CAD204520 (CAD2045202h) and hapsiga gin (Thapsiga gin200nM) on he same ca diac unc ions. G aph ba s: mean ± SD o he six CAD204520- ea ed ca diomyocy e g oups and mean ± SD o he wo hapsiga gin- ea ed ca diomyocy e g oups. S a is ical signi icance compa ing CAD204520- ea ed cells 98 e sus hapsiga gin- ea ed cells (**p ≤ 0.001, *p ≤ 0.05) was de e mined by a non- pa ame ic es (Mann-Whi ney). D) E ec o daily 30 mg/kg adminis a ion o CAD204520 on body weigh . E o ba s deno e he mean ± SD o six eplica es ( h ee male and h ee emale mice). S a is ical signi icance (n.s.) was de e mined by a wo-way ANOVA analysis. E) E ec o CAD204520 on T-ALL leukemia bu den in an SKW-3/KE-37-xenog a ed mu ine model. An i-leukemic ac i i y o CAD204520 assessed by measu ing hCD45+ cells a e 4 days o CAD204520 ea men (45 mg/kg/OS BID) o ehicle (Tween 80 0.5%, w/ , and hyd oxyp opyl-me hylcellulose [HPMC] 1.0%, w/ ). Rep esen a i e do plo showing he e ec o CAD204520 on T-ALL g ow h in an SKW-3/KE-37 mu ine model. A minimum o 20,000 e en s was collec ed o each condi ion. F) Immunohis ochemical analysis o he spleen in an SKW-3/KE-37-xenog a ed mu ine model ea ed wi h CAD204520 45 mg/kg o ehicle o 4 days. The spleens o all mice we e examined; ep esen a i e esul s o one con ol animal and one CAD204520- ea ed animal a e shown. Fo malin- ixed, pa a in-embedded issue sec ions we e s ained wi h hCD45. Scale ba s, 20 μm. G) Rep esen a i e images o hema oxylin/eosin-s ained sec ions o he le en icle om SKW-3/KE-37 xenog a ea ed wi h CAD204520 45 mg/kg o ehicle. CAD204520 ea men did no a ec he g oss s uc u al componen s o he myoca dium o induce ocal a eas o damage. Well-aligned myo ibe s in he absence o myocy oly ic nec osis o in e s i ial in lamma o y in il a es a e shown a highe magni ica ion (inse ). Scale ba s, 0.2 mm (low magni ica ion) and 0.05 mm (high magni ica ion; inse s). 99 H) Rep esen a i e images o hema oxylin/eosin-s ained his ological sec ions o he small in es ines om SKW-3/KE-37 xenog a ea ed wi h CAD204520 45 mg/kg o ehicle. Compa ed wi h con ols, in es inal illi and c yp s appea o be well p ese ed in CAD204520- ea ed animals. A highe magni ica ion (inse ), no mo phological changes in goble cells and en e ocy es we e obse ed in CAD204520- ea ed mice. Scale ba s, 0.2 mm (low magni ica ion) and 0.05 mm (high magni ica ion; inse s). I) E ec o CAD204520 on cell blood coun WBC, hemoglobin, and pla ele s in an SKW- 3/KE-37 mu ine model a e 4 days o CAD204520 ea men (45 mg/kg/OS BID) o ehicle (Tween 80 0.5%, w/ , and HPMC 1.0%, w/ ). E o ba s deno e he mean ± SD o eigh CAD204520- ea ed animals o he mean ± SD o eigh eplica es ehicle- ea ed mice. S a is ical signi icance o ea ed e sus ehicle (n.s.) was de e mined by non-pa ame ic es (Mann-Whi ney). Figu e 11. Resis ance o SERCA inhibi o s sensi ize T-ALL cells o glucoco icoids A) Small molecule iabili y sc een esul s o EU-OPENSCREEN (Eu opean Chemical Biology Lib a y) in ALL/SIL (blue lines) and ALL/SIL R ( ed lines) cell lines. All molecules we e es ed a 100 nM concen a ion. Rada plo shows he e ec o indi idual d ugs. F om he le : small molecules ac i e ( iabili y < 50%) on ei he ALL/SIL R, bo h cell lines o ALL/SIL only. B) Rada plo s epo ing d ug sc eening iabili y esul s o compounds ac i e on NR3C ecep o s in ALL/SIL (blue lines) and ALL/SIL R ( ed lines). All d ugs we e es ed a 100 nM. Each subg oup o NR3C ecep o is indica ed wi h a di e en colo . 100 C) Le : Wes e n blo ing showing exp ession o No ch1 pa hway and o al glucoco icoids ecep o (GR) in T-ALL cell lines. Righ : Real- ime PCR esul s compa ing he old change in gene exp ession o glucoco icoids ecep o iso o ms in ALL/SIL and ALL/SIL R cell lines in basal condi ions. The y axis ep esen s he old change be ween each condi ion and β- ac in exp ession as an in e nal con ol. E o ba deno es he mean ± SD o a minimum o h ee eplica es. S a is ical signi icance among g oups (****p < 0.0001) was de e mined by one-way ANOVA. D) E ec s o glucoco icoids on cell iabili y a e 72 hou s o ea men s in T-ALL cell lines. E o ba s deno e ± SD o 2 eplica es. E) E ec s o glucoco icoids on cell iabili y a e 72 hou s o ea men s in T-ALL cell lines in basal condi ions and a e a escue wi h ALL/SIL R medium. E o ba s deno e ± SD o 2 eplica es. Black: escue condi ions; pink: basal condi ions. F) E ec s o glucoco icoids on cell iabili y in ALL/SIL and ALL/SIL R T-ALL cell lines wi h he ollowing condi ions: a e 72 hou s o ea men s, a e 72 hou s o ea men s in combina ion wi h hapsiga gin 10 nM; a e a p e- ea men wi h RU486 1 μM and hapsiga gin 10 nM o 24 hou s and a ollowing ea men wi h glucoco icoids o 72 hou s. E o ba s deno e ± SD o 2 eplica es. G) Combina ion index (le ) and su ace plo s analysis ( igh ) o ALL/SIL and ALL/SIL R T- ALL cell lines ea ed wi h ehicle, CAD204520, lu icasone, o CAD204520 plus lu icasone. Each poin means an independen measu emen ep esen a i e o wo biological eplica es. Plo s we e gene a ed using Combene i sc ip by MATLAB R2021, which ep esen s he Loewe (dose-e ec based app oach) analysis. A colo scale ba ep esen s he le el o d ugs an agonism o syne gism. 101 Figu e S1 (Rela ed o Figu e 4 and Table S1): Syn hesis Rou e, Ac i i y and Binding Mode o CAD204520. A) Chemical s uc u e o he ini ial hi compound: 2-(2-py idyl)-6-( i luo ome hoxy)-1H- indole. B) Schema ic ep esen a ion o medicinal chemis y op imiza ion. R1 and R2 subs i u ions a e indica ed. C) Syn hesis ou e o CAD204520 (4-[2-[2-[3-p opyl-6-( i luo ome hoxy)-1H-indol-2-yl]-1- pipe idyl]e hyl]mo pholine). Syn he ic ou e (a) o (e) is depic ed and desc ibed in he me hods sec ion. D) De e mina ion o he p o ein ATP hyd olysis ac i i y in he p esence o compound CAD204520 a pH 7. The igu e displays ATPase ac i i y de e mined by measu ing he amoun o libe a ed phospha e om ATP hyd olysis. Da a is p esen ed as a i ed cu e which has been no malized o he maximal enzyme ac i i y wi h sub ac ion o backg ound signal om spon aneous hyd olysis o ATP. E o ba s deno e he mean ± SD (s anda d de ia ion) o 3 eplica es. S a is ical signi icance (**P ≤ 0.01; ***P ≤ 0.001) was de e mined by wo-way ANOVA using Bon e oni’s co ec ion o mul iple compa ison es ing. E) Binding si es o CAD204520 and hapsiga gin. Supe posi ion o he SERCA-CAD204520 complex wi h SERCA- hapsiga gin (PDB ID: 2AGV). The binding si es a e bo h in he ansmemb ane egion, sepa a ed by ansmemb ane helix M3. Thapsiga gin is shown as cyan su ace ep esen a ion. 102 F) Supe posi ion o SERCA-CAD204520 wi h SERCA-CPA (PDB ID 3FGO), iewed oughly along he memb ane no mal. CAD204520 and CPA a e shown as o ange and g een s icks, espec i ely. G) Supe posi ion o SERCA-CAD204520 wi h SERCA-DBHQ (PDB ID 2AGV) iewed oughly along he memb ane no mal. CAD204520 and DBHQ a e shown as o ange and ligh blue, espec i ely. H) Supe posi ion o SERCA-CAD204520 wi h SERCA-Cpd7 (PDB ID 5NCQ), iewed along he memb ane plane. CAD204520 and Cpd7 a e shown as o ange and magen a s icks, espec i ely. I) Binding si es o CAD204520 and hapsiga gin, as seen oughly along he memb ane no mal. Supe posi ion o SERCA bound o CAD204520 (ligh blue ca oon and o ange s icks, espec i ely) wi h SERCA bound o hapsiga gin (g ey ca oon and s icks, espec i ely). Glycine257, which is mu a ed o aline in he hapsiga gin esis an mu an , is indica ed by a ed sphe e. In he SERCA- hapsiga gin complex, Phe256 has unde gone a displacemen ha is likely o be impai ed by a aline esidue in posi ion 257. Figu e S2 (Rela ed o Figu e 5): Iden i ica ion o a hapsiga gin- esis an T-ALL cell line. A) E ec s o Thapsiga gin (le ) and CAD204520 ( igh ) on cell iabili y a e 72 hou s o ea men s in ALL/SIL and ALL/SIL hapsiga gin- esis an cell lines. E o ba s deno e ± SD o a minimum o 2 eplica es. B) Wes e n blo showing he exp ession o SERCA2 and SERCA3 in naï e and esis an ALL/SIL. β-ac in was used as a loading con ol. 103 C) Ph ed-scale analysis o exonic single nucleo ide a ia ion (SNV) occu ing in he ALL/SIL hapsiga gin esis an cell line. Inse shows numbe (N.) o a ia ion (SNV) occu ing pe ch omosome. D) Thapsiga gin esis ance mu a ion ho spo egion on helix M3. Thapsiga gin and SERCA esidues 254-260 a e shown in s ick ep esen a ion and colo ed cyan and ed, espec i ely. E) Su ace plo s analysis o ALL/SIL, DND41 and RPMI-8402 T-ALL cell lines and a p ima y NOTCH1-mu a ed T-ALL sample ea ed ehicle, CAD204520, cyclopiazonic acid, o CAD204520 plus cyclopiazonic acid. Each poin ep esen s an independen measu emen ep esen a i e o h ee biological eplica es. Plo s we e gene a ed using Combene i sc ip by MATLAB R2018 and ep esen he Loewe (dose-e ec based app oach) analysis. A colo scale ba ep esen s le el o d ug an agonism o syne gism. Figu e S3 (Rela ed o Figu e 6): E ec o CAD204520 on NOTCH1 mu a ed and wild ype T-ALL and MCL cell lines. A) Table ep esen ing NOTCH1 mu a ional s a us in T-ALL and MCL lines. B) Sca e do plo ep esen ing IC50 [μM] o CAD204520 in NOTCH1 mu a ed (n = 5) o NOTCH1 WT (n = 3) T-ALL o in NOTCH1 mu a ed (n = 1) o NOTCH1 WT (n = 3) MCL (shown in C) cell line. S a is ical signi icance (*P ≤ 0.05) was de e mined by a non-pa ame ic - es (Mann-Whi ney). D) E ec o CAD204520 ea men s on cycling MAVER-1 and MINO cells. Pe cen age o DNA con en ollowing ou days o ea men wi h he indica ed concen a ions o CAD204520 on each cell cycle phase is indica ed. A minimum o 20,000 e en s was collec ed o each condi ion. MOLT16 cell line (8;14)(q24;q11) SKW-3/KE-37 cell line (8;14)(q24;q11) CAD240520 0.0 0.5 1.0 1.5 F ac ion o Cell Ali e 5 * 2.5 DMSO CAD204520 [ µM ] SKW-3/KE-37 SKW-3/KE-37 MOLT16 MOLT16 BA CAD204520 [ µM ] *** 10.00 3.33 1.11 0.37 0.01 DMSO *** 30.00 *** 0 50 100 150 SKW-3/KE-37 MOLT16 200 200 400 600 800 1000 1200 CD SKW-3/KE-37MOLT16 2 0.5 DMSO 2 0.5 HSP90 FL-NOTCH1 TM-NOTCH1 DMSO CAD204520 [ µ M] CAD204520 [ µ M] Luminescence CAS/ATP Fold Inc ease CAD204520 [4 µM ] CAD204520 [8 µM ] T-ALL-mNOTCH1 B-ALL #1 #2 #1 #2 #1 #2 #1 #2 **** **** **** *** **** ** * **** 0.0 0.5 1.0 1.5 F ac ion o Cell Ali e A ea Unde he Cu e *** 0 50 100 150 Lymphocy e T-ALL FE Figu e 8 -dl/d max (µm/s) % o a ia ion Max Ra e o Sho ening (-dl/d max(µm/s)) Max Ra e o Re-Lengh ening (-dl/d max(µm/s)) F ac ion o Sho ening (FS(%)) 0 20 40 60 80 100 ** A 0 20 40 60 80 100 +dl/d max (µm/s) % o a ia ion ** B Con ol CAD204520 5 µ M 0 20 40 60 80 100 FS % o a ia ion CAD204520 5 µM Thapsiga gin 0.2 µM * Con ol Thapsiga gin 0.2 µ M C Days 0 4 8 12 16 20 0 10 20 30 Vehicle CAD204520 30 mg/Kg Body Weigh (g ) D E CD45-PE-A subse 5.64% 50K 100K 150K CAD204520 45 mg/Kg Vehicle CD45-PE-A subse 40.9% 50K 100K 150K 0 -10 3 10 3 10 4 10 5 FSC-A CD45-PE-A n.s. CAD204520 45 mg/Kg Vehicle 12 14 16 18 HGB (g/dL) n.s. Vehicle 700 800 900 1000 1100 1200 PLT (/ l) µ Vehicle n.s. CAD204520 45 mg/Kg 0 2000 4000 6000 WBC (/ l) µ CAD204520 45 mg/Kg I Figu e 10 Vehicle CAD204520 45 mg/Kg Vehicle CAD204520 45 mg/Kg Vehicle CAD204520 45 mg/Kg HF G 0 2 4 6 0 2 4 6 0 5 10 15 0 2 4 6 0 2 4 6 0 5 10 15 126.80 ±14.94 22.56 ±5.48 30.14 ±5.61 0.20CAD204522 37.68 ±6.22 20.09 ±2.76 58.86 ±6.50 0.21 CAD307496 21.81 ±4.50 0.28 ±0.07 7.75 ±1.68 0.26CAD204521 >333 0.04 ±0.01 1.56 ±0.45 0.25 CAD204630 71.25±18.00 3.16 ±0.41 18.00 ±3.25 0.21 CAD204631 9.74 ±1.67 0.39 ±0.20 1.03 ±0.22 0.27 CAD305666 84.22±24.37 0.01 ±0.02 0.55 ±0.12 0.28CAD204519 26.90 ±2.98 8.30 ±0.95 0.34 ±0.03 0.29CAD204520 7.75 ±2.69 0.59 ±0.17 0.32 ±0.19 0.22 CAD306749 16.80 ±2.68 1.21 ±0.37 2.62 ±0.83 0.22 CAD306750 Table S1