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 Coordinatore: Chiar.mo Prof. Nicola Sverzellati Tutore: Chiar.mo Prof. Giovanni Roti Co-Tutore: Chiar.mo Prof. Pieter Van Vlierberghe Dottorando: Dr. Luca Pagliaro Anni Accademici 2019/2020 – 2021/2022 1 ABSTRACT The identification of SERCA (sarco/endoplasmic reticulum calcium ATPase) as a target for modulating gain-of-function NOTCH1 mutations in Notch dependent cancers has spurred the development of this compound class for cancer therapeutics. SERCA plays a critical role in Ca2+ regulation particularly in myocytes, thus inhibiting it may increase the risk of heart failure, limiting the development of this compound class for cancer therapeutics. Considering this toxicity challenge, discovery and early optimization of small molecules with better druglike properties and reduced off-target toxicity is warranted. Despite the innate toxicity challenge associated with SERCA inhibition, we identified CAD204520, a small molecule with better drug-like properties and reduced off-target Ca2+ toxicity compared with the SERCA inhibitor thapsigargin. Similar to the SERCA inhibitor thapsigargin, CAD204520 inhibits Notch signaling in T-ALL leukemia cells causing a cell cycle arrest and induction of apoptosis. CAD204520 also preferentially targets mutated over wild type NOTCH1 proteins in T cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL), thereby overcoming the therapeutic limitation associated with the use of pan-Notch inhibitors. Remarkably, CAD204520 resulted in an effective treatment in a NOTCH1-mutated T-ALL in vivo model without causing overt Ca2+-related cardiac toxicity. To anticipate the potential mechanism of resistance to SERCA inhibitors, we generated a T-ALL cell line resistant to the effect of thapsigargin. A subsequent small molecule library screening identified glucocorticoids among the top hits in the resistant cell line. This effect is at least in part mediated by the specific upregulation of glucocorticoid receptor. Consequently, the association of SERCA inhibitors and glucocorticoids displayed a synergistic effect in multiple preclinical models. This study supports the development of SERCA inhibitors for Notchdependent cancers and extends their application to cases with isolated mutations in the 2 PEST degradation domain of NOTCH1, such as MCL or chronic lymphocytic leukemia (CLL). Furthermore, this study suggests that SERCA-Ca2+ modulation mediates glucocorticoid signaling and that innovative SI can pharmacologically modulate glucocorticoid resistance in T-ALL. 3 INDEX INTRODUCTION .................................................................................................................. 5 Targeting Notch Trafficking ............................................................................................... 5 SERCA ............................................................................................................................. 5 SERCA and Cancer ........................................................................................................ 11 SERCA and Notch .......................................................................................................... 13 AIM OF THE STUDY ......................................................................................................... 17 MATERIALS AND METHODS ........................................................................................... 19 Experimental Models and Subject Details ...................................................................... 19 Data and Code Availability .............................................................................................. 21 Preparation, Crystallization and Structure Determination of the SERCA-CAD204520 Complex .......................................................................................................................... 22 Synthesis Pathways ........................................................................................................ 23 ATPase Preparation ....................................................................................................... 31 ATP Hydrolysis Inhibition ................................................................................................ 32 Cell Viability, Apoptosis and DNA Content Assays ......................................................... 33 Cell Competition Assay ................................................................................................... 34 Compound Sources ........................................................................................................ 34 Compound Treatment of Cell Lines and Primary Cells ................................................... 34 Intracellular Calcium Measurement ................................................................................ 35 Western Blot ................................................................................................................... 36 Indirect Immunofluorescence Microscopy ...................................................................... 37 Real-time RT-PCR .......................................................................................................... 38 Whole Exome Sequencing ............................................................................................. 38 Virus Production and Transduction of T-ALL Cell Lines ................................................. 39 Cardiomyocyte Isolation and Treatment ......................................................................... 40 Intracellular ATP Content Detection ............................................................................... 42 In Vivo Studies ................................................................................................................ 42 Small molecule screening assay .................................................................................... 44 Quantification and Statistical Analysis ............................................................................ 44 RESULTS ........................................................................................................................... 45 4 Identification of CAD204520 as a Selective Ca2+ ATPase Inhibitor ................................ 45 CAD204520 Rescues T-ALL Cells from Thapsigargin Resistance ................................. 48 CAD204520 Suppresses Leukemia Growth in NOTCH1-Mutated T-ALL and MCL ....... 51 CAD204520 Suppresses Notch1 Signaling .................................................................... 51 CAD204520 Preferentially Inhibits NOTCH1-Mutated Cancers ..................................... 53 Consequences of Ca2+ Release upon CAD204520 Treatment ...................................... 54 Modeling Preclinical Toxicity and Efficacy of CAD204520 in a T-ALL Leukemia Model 56 Identification of glucocorticoids as potential rescue treatment in NOTCH1-mutated cell line carrying ATP2A2 mutation ....................................................................................... 59 ATP2A2 mutation induces upregulation of glucocorticoid receptor ................................ 60 SERCA inhibitors synergize with glucocorticoids with a better profile in ATP2A2 mutated cells ................................................................................................................................. 61 DISCUSSION ..................................................................................................................... 62 CONCLUSIONS ................................................................................................................. 68 BIBLIOGRAPHY ................................................................................................................ 70 FIGURE LEGENDS ........................................................................................................... 89 5 INTRODUCTION Targeting Notch Trafficking The Notch signaling pathway plays an important role in the pathogenesis of human cancers. Notch controls both cell-intrinsic and extrinsic circuits leading to tumor development, progression and response to therapy. Several therapeutic efforts have historically focused on modulating Notch signaling by using small molecules such as g-secretase inhibitors (GSI) or antibody-based strategies. However, these approaches have a poor therapeutic window - wild type vs mutant proteins - limiting their application in human diseases. This is not the case of small molecules targeting the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA). SERCA inhibition hijacks Notch trafficking and its activation, emerging as druggable approach for NOTCH-dependent cancers. SERCA SERCA proteins belong to the superfamily of active transporters known as P-type ATPases (E1/E2-type) located in the endoplasmic reticulum (ER). In 1993 Toyoshima and colleagues described the first complete structure of SERCA by cryo-electron microscopy (1). Subsequently, novel high-resolution techniques shaped the resolution of several crystallography structures of SERCA. These studies showed how ligands (e.g., vanadate, thapsigargin) bind SERCA, and what structural changes occur during the enzymatic catalytic cycle (2-6). The SERCA protein comprises ten helices (M1-M10), a small luminal tail and three cytoplasmic domains (A, actuator; N, nucleotide binding; P, phosphorylation). These modules mediate ATP hydrolysis, hydron (H+) and calcium (Ca2+) binding and their transport through ER membrane (7). ATP2A1 (16p11.2), ATP2A2 (12q24.11) and ATP2A3 (17p13.2) genes encode for SERCA1, 2 and 3 respectively (8, 9). Today, over 70 SERCA isoforms 6 resulting from alternative splicing are deposited in the Protein Data Bank database (www.rcsb.org). While these transcripts share up to 85% of sequence homology, differences in tissue distribution, Ca2+ binding affinity both in normal and cancer tissue are due to changes in the protein C-terminal region (10-15). SERCA proteins maintain intracellular Ca2+ homeostasis by pumping Ca2+ from cytosol into the ER (16). This adenosine triphosphate (ATP) (10) dependent catalytic cycle alternates different SERCA phosphorylated intermediates (E1P; E2P) with different conformation for high (E1-Micromolar-μM) or low (E2-Millimolar-mM) affinity for Ca2+: this cycle, named PostAlbers scheme by the first proponent, was originally applied to other types of ion pump, such as the Na+/K+-ATPase, and subsequently adapted to SERCA (17). The net balance of the ATP hydrolysis required to complete the cycle is of two Ca2+ ions transferred from the cytosol to the ER lumen, and two or three protons in the opposite direction towards the cytosol (18) (Figure 1). All SERCA isoforms present two Ca2+ binding sites (site I and II) between four transmembrane (TM) helices (M4, M5, M6 and M8), near the cytoplasmic side of the membrane. Site I contains five amino acids residues from three different transmembrane helices associated with two water molecules: Asn768 and Glu771 from M5, Thr799 and Asp800 from M6 and Glu908 from M8 (19). Site II is located on the cytoplasmic surface next to the site I and it is composed of Ile307 and Gly310 from M4 helix, that is partially unwound, and Asn796 and Asp800 from M6; both sites provide one to three chain of oxygen to the coordination (19). Interestingly, the binding of the first Ca2+ ion on site I acts in a cooperative way, bringing to a conformational change on the oxygen chains of site II and allowing an high affinity state for the second ion binding (16, 19, 20). 7 SERCA, is organized in functional domains that mediate ions binding, ATP hydrolysis and ions transport through membrane (7). The N domain is the largest one to accommodate through the binding at the Phe487 residue, the adenine ring of the ATP (19, 21, 22). The Ndomain is linked, through the Arg560 residue to the surrounding enzymatic P domain that contains the amino acid residues targeted by the phosphorylation. For example, phosphorylation of the Asp351 residue allows for a 25-30 Å rearrangements of the P-domainATP complex leading to generation of the high energy enzyme intermediate (16, 20, 22, 23). The A domain realizes the gating mechanism of calcium transport. The A domain is the smallest cytosolic domain and is situated near the M1, M2 and M3 helices with which is bound by flexible linkers allowing a better range of movement for ATP utilization. Similarly to other P-type ATPases, the A domain presents conserved protein motives such as the TGES motif, responsible for the dephosphorylation of the Asp351 residue during the catalytic cycle (16, 19, 20, 23, 24). The high-resolution crystal structures of the SERCA bound to Ca2+, thapsigargin, and vanadate provided the frame for the understanding of structural changes occurring during the catalytic cycle (19). Thapsigargin binds to SERCA fitting in a cavity composed by M3, M5 and M7 helices (25). The most important amino acid residue that interacts with Thapsigargin is Phe256 on M3 helix, assisted by Ile765 on M5 and Tyr837 on M7 (25, 26). Mutations affecting the residue Phe256 bring to thapsigargin resistance confirming the pivotal role of this amino acid in the inhibition of the enzyme, even if the reduction of sensitivity of SERCA caused by the mutation is different among the isoforms, with SERCA2b the least affected (14, 26). Other SERCA inhibitors, like cyclopiazonic acid (CPA) or 2,5-di-(t-butyl)-1,4-benzohydroquinone (DBHQ), bind the enzyme in a different pocket, between the transmembrane helices M1, M2, M3 and M4 (27, 28), identified as the entrance pathway of calcium ions from cytosol (29). 14 and in a subset of B-cell-like (activated B-cell, ABC) diffuse large B-cell lymphoma (94). Most of these mutations occur in the juxtamembrane heterodimerization (HD) domain, which hold together the ECD-N with TM-N, or in the PEST (rich in proline (P), glutamic acid (E), serine (S), and threonine (T)) degron domain. In T-ALL, HD and PEST mutations may cooccur while isolated PEST are common in CLL and MCL. Here, a premature stop codon (P2514fs*4) generally truncates the C-terminal PEST region of the protein. The PEST domain controls the degradation of activated NOTCH proteins, and its deletion results in a delayed protein half-life. In addition, the Notch signaling pathway is frequently activated in multiple types of solid tumors (95, 96), such as melanoma, colorectal carcinoma, and cholangiocarcinoma through mechanisms that differ from genetic variations (97, 98). Paradoxically, mutations that inactivate the Notch pathway have been described in several human cancers (96, 99) showing that, depending on the cellular context, Notch signaling can be oncogenic or tumor suppressive and suggesting that fine-tuned inhibition of Notch signaling could be useful in those situations where Notch is activated. The preponderance of oncogenic NOTCH1 mutations in T-ALL has prompted the search for effective anti-Notch1 therapeutics (75, 100). Because Notch activation relies on g-secretase mediated proteolysis, GSI had entered in clinical trials to treat relapsed T-ALL. However, first generations of GSIs were poorly tolerated because of on-target gastro-intestinal toxicity (101-103). As showed by Riccio and colleagues the toxic effect of GSIs are a consequence of lack of substrate specificity of these molecules resulting in the combined inhibition of wild type NOTCH1 and NOTCH2 in intestinal progenitor cells (104). Although few patients achieved a complete response, GSIs exhibited moderate clinical activity in some patients with solid tumor and leukemia (105). Recently, several studies demonstrated that combining 15 GSIs with chemotherapy or other targeted agents increases the anti-cancer effects of these drugs (106-109), supporting the development of innovative anti-Notch1 therapeutics. The rise of SERCA inhibitors for cancer therapeutics date back to 1960 when the National Cancer Institute (NCI) launched a program to identify compounds with antitumor activity from 35,000 plants extracts (110). Sesquiterpene lactone (SL) derivatives demonstrated an antiinflammatory and antitumor activity in several tumor types, like laryngeal carcinomas, uveal melanomas, pituitary macroadenomas, kidney, prostate cancer and hematological malignancies (111-116). Among others, thapsigargin, parthenolide and artemisinin were selected for their potency and initially used as tool compounds in different cancer models. Several SERCA inhibitors which differed in their source, chemical structure, potency and binding affinity to specific SERCA isoforms were subsequently developed (82). In 1999, Goran Periz and Mark E. Fortini described that the trafficking events leading to a correct NOTCH activation may be disrupted in the presence of a defective Ca2+-ATPase function in a Drosophila model. In this work, the authors demonstrated that loss-of-function alleles of the Drosophila SERCA homologous gene Ca-P60A alters proper synthesis, folding and trafficking of the NOTCH receptor in the ER/Golgi compartments (Figure 2). Consistently, in Drosophila S2 cultured lines, the treatment with general SERCA inhibitors such as thapsigargin and CPA primarily reduces the amount of NOTCH proteins that reach the cell surface (117). While extremely interesting, these observations were not confirmed in mammalian cells until, Giovanni Roti and Kimberly Stegmaier embarked on a large geneexpression based screening (GE-HTS) effort to identify inhibitors of oncogenic NOTCH1 signatures or enhancer of NOTCH1 HD mutant L1601P∆P activity in T-ALL. Among the top hits were the genes ATP2A2 and ATP2A3, and SERCA inhibitors such thapsigaricin (an analogue of thapsigargin) and CPA. Together with other ion flux modulators, SERCA 16 emerged as a novel potential therapeutic target in NOTCH1-associated cancers (75, 118). Furthermore, these data suggest the hypothesis that Notch signaling could be dysfunctional in several genetic disorders associated with loss of function ATPA1-3 mutations. However, thapsigargin binding to SERCA results in an increase in cytosolic Ca2+ concentration and a depletion of Ca2+ stored in the ER. Thus, the delivery of free thapsigargin to humans might cause cardiac toxicity due to a rapid calcium ion shift. To overcome this limitation in the past we generated a thapsigargin pro-drug taking advantage of the dependency of ALL on folic acid (FA) and, tagged folate to a permissive site on an active alcohol derivative of thapsigargin (8-O-debutanoylthapsigargin) via a cleavable ester linkage (JQ-FT) (119). However, an alternative approach to reducing the potential toxicity of thapsigargin is through the identification of SERCA inhibitors that retain the anti-Notch properties but lack Ca2+ related toxicities (Figure 3). 17 AIM OF THE STUDY Given its critical oncogenic role in several human cancers, Notch1 signaling has garnered increased attention as a potential therapeutic target. To date, several Notch inhibitors, including GSI, have shown therapeutic efficacy in preclinical cancer models. However, despite this promise, few of these candidates have been demonstrated to have a meaningful clinical benefit for patients, in part due to tissue-dependent on-target toxicities from the simultaneous repression of both mutant and wild type NOTCH proteins. The discovery of SERCA as actionable modulators of Notch1 suggested a new targeted approach to treat T-ALL. Uniquely among Notch modulators, SERCA inhibitors preferentially impair the clinically relevant class of oncogenic NOTCH1 mutants compared to wild type. Thus, the development of tolerated SERCA modulators may uncover a new therapeutic avenue for one of the most frequently mutated genes in human cancers. In this work, we identified a new series of P-type ATPase/SERCA inhibitors and characterized the effect of CAD204520 in NOTCH1-mutated T-ALL. We demonstrated that CAD204520 exhibits a reduced Ca2+ related off-target toxicity but retains anti-Notch1 and anti-leukemia capacity both in vitro and in vivo in NOTCH1-mutated T-ALL models. In addition, we extended the testing of CAD204520 in B-cell malignancies carrying clinically relevant PEST mutations and demonstrated, for the first time, the potential of SERCA inhibition as a therapeutic approach in these diseases. Furthermore, to anticipate the potential mechanism of resistance to SERCA inhibitors, we generated a T-ALL cell line (ALL/SIL) resistant (R) to the effect of thapsigargin by exposing the cells to increased concentrations of thapsigargin. Exome sequencing analysis identified a mutation (c.G770Tà p.G257V) in the ATP2A2 locus that impedes an efficient thapsigargin 18 binding to the protein, finally resulting in a diminished inhibitory effect. we then screened a small molecule library of nearly 2500 bioactive compounds (from the European Chemical Biology Library provided by EU-OPENSCREEN) in ALL/SIL and ALL/SIL R. Compound hits were flagged by their ability to preferentially inhibits ALL/SIL, ALL/SIL R, or both. Confirmatory experiments and pathway analysis were completed in multiple T-ALL preclinical models. 19 MATERIALS AND METHODS Experimental Models and Subject Details Animals NOD-scid IL2Rgammanull (NSG) mice (The Jackson labs, RRID: IMSR_JAX:005557) for efficacy studies were maintained in specific pathogen–free facilities at the ‘‘Preclinical Research Services Center’’ (Ce.Se.R.P) at the University of Perugia (08/2018-UT of 07/24/2018). Animal procedures were approved by the University of Perugia IACUC following the DL 26/2014 and 2010/63/EU guidelines for the protection of animals used for scientific purposes. Pharmacokinetics (PK) and tolerability studies were performed at Aurigene Discovery Technologies, India. In-house breed CD1 (ICR) or BALB/cAnNCr mice for pharmacokinetics (PK) and tolerability studies were maintained in individually ventilated cages at the Aurigene facility in Hyderabad in India. To assess effect of CAD204520 on the cardiac mechanics, cardiomyocytes were isolated from 12-14 week aged Wistar rats (Rattus norvegicus, Charles River Laboratory, RRID: RGD_13508588) of 362 ± 5 g in weight. Animals were housed in a temperature-controlled room (22–24°C), with a 12 hour light cycle (light on from 7.00 AM to 7.00 PM) with unrestricted food and water supply. Experiments were performed under the Veterinary Animal Care and Use Committee of the University of Parma-Italy and conform to the National Ethical Guidelines of the Italian Ministry of Health (Prot. N 614/2016-PR) and the Guide for the Care and Use of Laboratory Animals (National Institute of Health, Bethesda, MD, USA, revised 1996). Yeast Cells Saccharomyces cerevisiae RS72 yeast cells (ATCC #9763) for the biochemical ATPase assay were pre-cultured in 100 ml sterile SGAH medium (7.04 g/L yeast nitrogen base, 19.8 20 g/L galactose, 64 mg/L adenine, 64 mg/L histidine) for 3 days at 25°C and 150 rpm. The pre-culture was transferred to 500 mL sterile SGAH medium and further incubated for 3 to 4 days. 100 mL from the cell culture was transferred to 1 L YPAD medium (10 g/L yeast extract, 20 g/L bacto-peptone, 20 g/L glucose, 20 mg/L adenine) and incubated at 25°C for 18-20 hours. Cell Lines Human cell lines DND41 (source: male), MOLT16 (source: female), PF382 (source: female), RPMI-8402 (source: female), SKW-3/KE-37 (source: male), JURKAT (source: male), CTV1 (source: male), HBS2 (source: male), Loucy (source: female) and PEER (source: female), REC-1 (source: male), Mino (source: female), were purchased from Leibniz-Institut DSMZGerman collection of microorganisms and cell cultures (Germany); the identity of ALL/SIL (source: male) was confirmed by PCR sequencing for known NOTCH1 mutations and short tandem repeat (STR) loci profiling and they were kindly provided by Stegmaier laboratory. MAVER-1 (source: male) cells were a gift from the Muraro laboratory (C.R.O. National Cancer Institute, Aviano, Italy). Cells were cultured in RPMI 1640 (Fisher Scientific, Waltham MA, USA #MT10040CV) with 10% or 20% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham MA, USA, #10270-106) and 1% penicillin-streptomycin (Fisher Scientific, Waltham MA, #3MT30002CI) and incubated at 37°C with 5% CO2. 293T (source: human primary embryonal kidney cell line 293 (ACC 305)) cells were purchased LeibnizInstitut DSMZ-German collection of microorganisms and cell cultures (Germany) and cultured in DMEM (Fisher Scientific, Waltham MA, USA #11965-084) with 10% FBS and 1% penicillin-streptomycin and incubated at 37°C with 5% CO2. HL-1 cardiac muscle cell line was a kind gift from the Miragoli laboratory (University of Parma, Parma, Italy). HL-1 cells were plated on a gelatin layer derived from bovine skin/fibronectin (1mg/ml) (Sigma-Aldrich, St. Louis, MO, USA, #G9391 and #F-1141) coated T25 flask and cultured in Claycomb 21 medium (Sigma-Aldrich, St. Louis, MO, USA, #51800C) with 10% FBS, 1% penicillinstreptomycin, 2mM L-Glutamine (Thermo Fisher Scientific, Waltham MA, USA, Waltham MA, USA, #25030081), 0,1mM Norepinephrine [(±)- Arterenol] plus L-Ascorbic acid, sodium salt (Sigma-Aldrich, St. Louis, MO, USA, #A0937 and #A7506) and incubated at 37°C with 5% CO2. Cytogenetic, FISH and mutation analysis was completed according to validated methods as previously described (120, 121). Primary Samples T-ALL lymphoblasts were obtained from patients with leukemia under an approved protocol at the Parma University Hospital (n.18249/18/05/2017) and according to the declaration of Helsinki guidelines for the protection of human rights. Peripheral blood (PB), and bone marrow (BM) samples were collected at the time of diagnosis, and we retained samples with blasts >85%. Mononuclear cells were isolated by density gradient centrifugation using LSMlymphocyte separation medium (CappelTM MP Biomedicals, LLC, Ohio, USA #50494). Lymphocytes were isolated from peripheral blood mononuclear cell (PBMC) by using a CliniMACS Prodigy (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured for a short time using the same growth conditions described above. Data and Code Availability The model and structure factors for the SERCA-CAD204520 complex structure reported in this paper have been deposited in the Protein Data Bank: PDB: 6YAA. 22 Preparation, Crystallization and Structure Determination of the SERCA-CAD204520 Complex Rabbit sarcoplasmic reticulum (SR) membranes containing SERCA were prepared from rabbit hind leg muscle as previously described (122). Briefly, muscle tissue was dissected and minced in 10 mM KCl, 2.5 mM K2HPO4, 2.5 mM KH2PO4, 2 mM EDTA, followed by centrifugation at 4°C for 20 min and 6.400 x g, supernatant filtered and spun at 9.700 x g for 20 min. SR membranes were sedimented by a 60 min centrifugation at 47.800 x g at 4°C. Membranes were homogenised and washed successively with buffers B (1 M sucrose, 50 mM KCl), C (1 M KCl, 3.4 mM MgATP pH 7.0), D (50 mM KCl), and E (0.3 M sucrose, 5 mM Hepes pH 7.4). Finally, washed membranes were extracted twice with extraction buffer (0.3 M sucrose, 0.5 M KCl, 1 mM EDTA, 10 mM Tris, 0.01 mM CaCl2, 1.25 mM MgCl2, pH 7.9, 0.5 mg/ml DOC, 0.5 mg/ml DTT) followed by centrifugation at 4°C and 181.000 x g for 75 min. The pellet was then washed with 5 mM TAPS pH 7.5, 0.3 M sucrose, 0.5 M KCl, 0.5 mM MgCl2, 10 µM CaCl2, and finally resuspended and flash frozen in buffer E. SERCA membranes were resuspended and gently homogenized in 100 mM MOPS-Tris pH 6.8, 80 mM KCl, 3 mM MgCl2, 4 mM EGTA and 20% (v/v) glycerol. CAD204520 was added to the membrane preparation at a final concentration of 0.5 mM and incubated overnight at 4°C. The following day, 0.4 mM TNPATP were added and incubated for 15 minutes (min) prior to the solubilization of the protein with C12E8 at a detergent/protein ratio of 1.5:1 (w/w). After 10 min incubation and centrifugation (TLA-100.3 rotor, 50,000 rpm, 30 min, 4°C), the concentration of solubilized protein was usually 10–12 mg/mL. Co-crystallization of SERCA with CAD204520 was carried out using hanging drop equilibration at room temperature (RT) with protein/buffer in a 1:1 ratio. The best diffracting crystals were obtained with crystallization buffer consisting of 10% glycerol, 14% PEG 6000, 100 mM NaCl and 6% MPD. Data were collected at 100 K and a wavelength of 0.976 A at beam line I03 at the 23 Diamond Light Source (DLS) in Didcot, UK. The data were processed using XDS (123) and AIMLESS (124) and the structure was determined by molecular replacement in PHASER (125) using a SERCA crystal structure with matching space group (pdb: 4UU0) (126). PHENIX (127) was used for refinement, ligand fitting and model validation and COOT for model building (128). Figures were prepared with Pymol (Molecular Graphics System, Version 2.0 Schro  dinger, LLC). Synthesis Pathways CAD307496 (2-[1-[3-(3-pyridyl)propyl]-2-piperidyl]-6-(trifluoromethoxy)-1H-indole) was prepared through the following intermediates: a) Intermediate 2-[2-(2-pyridyl)ethynyl]-5-(trifluoromethoxy)aniline: 2-Bromo-5- (trifluoromethoxy)aniline (6.21g, 24,2 mM), 2-ethynylpyridine (2.50g, 24.2 mM) and potassium carbonate (8.38g, 60.6 mM) in NMP (50 mL) were degassed with argon. Pd(DtBPF)Cl2 (474mg, 0.73 mM ) was added and the reaction heated under argon to 120°C for 1 hour 45 min. The reaction cooled and diluted with water, extracted with 1:1 cHexane/EtOAc (3x 150 ml), washing each extract well with water. The combined extracts were dried, evaporated, and columned on 100 g SNAP cartridge eluting with 0-60% EtOAc/cHexane, using 20 column volumes to yield 750 mg of title compound as a brown solid. This was used without further purification. b) Intermediate 2-(2-pyridyl)-6-(trifluoromethoxy)-1H-indole: To 2-[2-(2-pyridyl)ethynyl]- 5-(trifluoromethoxy)aniline (950mg, 3.41 mM) was added potassium 2-methylpropan2-olate (383.13 mg, 3.41 mM) in DMF (50 mL) and the mixture was stirred at RT overnight. The reaction was quenched with 0.4 mL HOAc concentrated in vacuo, dissolved in diethyl ether and washed with sat. NaHCO3 and water. The organic 30 b) Intermediate 2-[2-(2-piperidyl)-6-(trifluoromethoxy)-1H-indol-3-yl]ethanol was prepared similar to intermediate B (step (b) for CAD204631). Yield 45%. c) Intermediate 2-[2-[1-[(4-bromophenyl)methyl]-2-piperidyl]-6-(trifluoromethoxy)-1Hindol-3-yl]ethanol was obtained using Intermediate 2-[2-(2-piperidyl)-6- (trifluoromethoxy)-1H-indol-3-yl]ethanol and commercially available 4bromobenzaldehyde (CAS 1122-91-4) and a procedure similar to CAD204521. Yield 87%. QC-LCMS (ESI): (m/z) (M+H)+ = 497.1 (M-H)- = 495.1. CAD204630 (2-[1-[(4-bromophenyl)methyl]-2-piperidyl]-3-isopentyl-6-(trifluoromethoxy)-1H-indole hydrochloride): a) Intermediate 3-isopentyl-2-(2-pyridyl)-6-(trifluoromethoxy)-1H-indole: By a method similar to that of CAD204631 step (a) above using commercially available 2-Bromo5-(trifluoromethoxy)aniline (CAS 887267-47-2) and 2-(5-methylhex-1-ynyl)pyridine. Yield 60%. b) Intermediate 3-isopentyl-2-(2-piperidyl)-6-(trifluoromethoxy)-1H-indole: By a method similar to intermediate B (step b for CAD204631). Yield 56%. c) 2-[1-[(4-bromophenyl)methyl]-2-piperidyl]-3-isopentyl-6-(trifluoromethoxy)-1H-indole hydrochloride: By a method similarly to that of CAD204521 employing commercially available 4-bromobenzaldehyde (CAS 1122-91-4). Yield 62%. QC-LCMS (ESI): (m/z) (M+H)+ = 523.1 (M-H)- = 521.1. Note: Many of the compounds are racemates i.e. of enantiomers or diastereomers. The pure enantiomer was not isolated 31 ATPase Preparation Heat competent Saccharomyces cerevisiae RS72 yeast cells (129) were transformed using a lithium acetate, single-stranded carrier DNA/polyethylene glycol method and with a yeast multicopy vector (130) containing the full-length cDNA of the S. cerevisiae plasma membrane H+-ATPase isoform PMA1 under control of the PMA1 promoter. Transformed yeast cells were pre-cultured in 100 mL sterile SGAH medium (7.04 g/L yeast nitrogen base, 19.8 g/L galactose, 64 mg/L adenine, 64 mg/L histidine) for 3 days at 25°C and 150 rpm. The pre-culture was transferred to 500 mL sterile SGAH medium and further incubated for 3 to 4 days. 100 mL from the cell culture was transferred to 1 L YPAD medium (10 g/L yeast extract, 20 g/L bacto-peptone, 20 g/L glucose, 20 mg/L adenine) and incubated at 25°C for 18-20 hours. Recombinant yeast was harvested by 2-3 min of centrifugation at 3.000 x g and 4°C, followed by 2 times wash in milli-Q water. Harvested cells were incubated in 10% glucose for 10 min, on a shaking table, and centrifuged at 3.000 x g and 4°C. Cells were resuspended in homogenisation buffer (50 g/L glucose, 28.3% glycerol, 0.1 M Tris-HCl pH 7.25, 10 mM EDTA pH 8.0, 50 mM KCl, 1mM DTT, 200 µM PMSF, 2 µg/ml Pepstatin A), and disrupted with 165 g glass beads (500 µm) by runs in a BeadBeater (Biospec). The disrupted cells were centrifuged at 4°C for 5 and 15 min at 1.400 x g and 12.000 x g, respectively. The supernatant was collected and centrifuged at 251.000 x g for 1 h with 112 µM phenylmethylsulfonyl fluoride (PMSF) and 1.1 µg/mL Pepstatin A. The resulting pellet was re-suspended in GTEK20 buffer (20% glycerol, 10 mM Tris-HCl pH 7.25, 25 mM KCl, 0.5 mM EDTA pH 8.0, 1 mM DTT, 0.2 mM PMSF, 2 µg/ml Pepstatin A) and centrifuged for 45 min at 251.000 x g and 4°C. Pellet was then re-suspended in STKED20 buffer (200 g/L sucrose, 40 g/L glucose, 50 mM Tris-HCl pH 7.25, 50mM KCl, 1 mM EDTA pH 8.0, 1 mM DTT, 0.2 mM PMSF, 2 µg/ml Pepstatin A), homogenised and diluted with STKED20 buffer. The plasma membranes were recovered at the interface of a 43%/53% (wt/wt) step sucrose 32 gradient containing sucrose in 50 mM Tris-HCl pH 7.25, 50 mM KCl, 1 mM EDTA, 1 mM DTT. Centrifugation was done for 16 h at 154.000 x g and 4°C. The plasma membrane fraction was collected and diluted with GTEK20 buffer and centrifuged for 1 hour at 274.000 x g. Pellet was collected and homogenised in GTEK20 buffer and stored at -80°C. Sarco/Endoplasmic reticulum (SR) Ca2+-ATPase was provided in SR membranes purified by extraction with a low concentration of deoxycholate (DOC) as described above. The pig kidney Na+/K+-ATPase purification included a mild SDS treatment of isolated microsomes followed by a washing step and was kindly performed by Natalya Fedosova, Aarhus University and prepared as described in (131). In brief, pieces of outer medulla were extracted and cut in pieces and further suspended and homogenized in ISE-buffer (25 mM imidazole, 250 mM sucrose, 1 mM EDTA pH 7.4). Microsomes were isolated by differential centrifugations. The final pellet was suspended and homogenized in ISE-buffer and stored at -20°C. ATP Hydrolysis Inhibition ATPase activity was determined by measuring the amount of liberated phosphate from ATP hydrolysis. The ATPase assay was performed in 96 well plates in a final reaction volume of 60 µL. 0.1-0.2 µg/well of DOC extracted SERCA membrane or the Na+/K+-ATPase was used, while 1-2.5 µg/well was used of the Pma1 membrane preparation. Reactions including protein membrane preparation and exogenously added compounds in a ½ log dilution concentration range from 333 µM or 166 µM to 0.005 µM. We conducted the enzymatic reactions in the following buffers; Pma1 buffer: 17.5 mM MOPS-NaOH pH 7,7 mM MgSO4, 44 mM KNO3 (vacuolar ATPase inhibitor), 22 mM NaN3 (mitochondrial ATPase inhibitor), 0.22 mM Na2MoO4 (acid phosphatase inhibitor); SERCA buffer: 9 mM MOPS-NaOH pH 7, 33 2.7 mM MgCl2, 0.1 mM CaCl2 and 72 mM KCl. Na+/K+-ATPase buffer: 30 mM MOPS-NaOH pH 7, 40 mM NaCl, 4 mM MgCl2 and 20 mM KCl. Reactions were started by the addition of Na-ATP to a final concentration of 2.5 mM (Pma1 and Na+/K+-ATPase) or 5 mM (SERCA), followed by 30 min incubation at 30°C. The amount of liberated phosphate was determined calorimetrically after addition of STOP solution (mixture of L-ascorbic acid, ammonium heptamolybdate tetrahydrate, and HCl to give final concentrations of 65 mM, 2.2 mM, and 189 mM, respectively) with 5 min incubation at RT followed by addition of arsenite solution (mixture of NaAsO2, sodium citrate dihydrate, and acetic acid to give final concentrations of 3.1 mM, 28 mM, and 141 mM, respectively). We measured absorption at 860 nm after additional 30 min incubation at RT. Cell Viability, Apoptosis and DNA Content Assays ATP-based cell viability was determined using the CellTiter-Glo viability assay (Promega Corporation, Madison, WI, USA #G7573) and luminescence was measured using a Victor X4 (Perkin Elmer, Waltham, MA, USA). Apoptotic rate was quantified by staining cells with Annexin V and propidium iodide using a flow-cytometry commercial kit (eBioscience™ Annexin V Apoptosis Detection Kit APC, Waltham MA, USA, # 88-8007-74). Cells were analyzed by flow cytometry with a FACScan flow cytometer (Beckman Culture-Cytomics FC 500, Life Sciences Division, Indianapolis, USA) and FlowJo V10 (Tree Star LLC, Ashland, OR, USA) analytical software. Cellular DNA content was assessed by staining with propidium iodide (50 g/mL) and analyzed by flow cytometry. At least 20,000 events were acquired and all determinations were replicated at least twice. 34 Cell Competition Assay SKW-3/KE-37-GFP and MOLT16 were co-cultured at 1:1 ratio in RPMI 1640, 10% FBS, 1% P/S medium. 1 x 106 cells per condition were treated with CAD204520 at the following concentrations 2.5 and 5 µM and DMSO at 0.005% and 0.01% respectively and incubated at 37°C. After 72 hours, T-ALL cells were washed in PBS, and stained with a LIVE/DEAD Fixable Far Red Dead Cell Stain (Invitrogen, Life Technologies, Carlsbad, CA, USA, #L34973) for 30 min. Fluorescent signal was assessed by flow-cytometry [Beckman CultureCytomics FC 500 (Life Sciences Division, Indianapolis, USA) and FlowJo V10 (Tree Star LLC, Ashland, OR, USA) analytical software]. A minimum of 20,000 events was collected for each biological sample. Experiments are representative of two independent experiments. Compound Sources We obtained the compounds for this study from the following sources: DAPT (N-[N-(3,5difluorophenacetyl)-1-alanyl]-(S)-phenylglycine) (Selleckchem, Houston, TX USA, #S2215), thapsigargin (Enzo Biochem, Inc., USA #BML-PE180-0005), dexamethasone, clobetasol propionate, fluticasone propionate and RU486 were purchased from MedChemExpress EU (MCE) (MedChemTronica, Sweden, #HY-14648; #HY-13600; #HY-B0154; #HY-13683). Compound Treatment of Cell Lines and Primary Cells Cells were seeded in 384-well plates (Corning Life Sciences Plastic, Bedford MA, USA, #3570) at the final concentration of 0.02 x 106/mL per condition. Small molecules were added with a nanometric dispenser Tecan D300e (Tecan Trading AG, Switzerland), and cellular viability was assessed after 72 hours of drug treatment using a CellTiter-Glo ATP 35 assay (Promega Corporation, Madison, WI, USA, #G7573). IC50 and the area under the curve (AUC) were calculated using GraphPad Prism software (La Jolla, CA, USA). Intracellular Calcium Measurement Cytosolic Ca2+ concentration was measured using the Indo-1 AM probe (ThermoFisher Scientific, Waltham MA, USA, #I1223). Cells were washed twice with a calcium free solution (D-PBS Life Technologies, Carlsbad, CA, USA, #10010015) and loaded at 37°C in 5% CO2 for 30 min with 5 µM of Indo-1 AM. Then, cells were washed twice with D-PBS and equilibrated in RPMI 1640 (Thermo Fisher Scientific, Waltham MA, USA, Waltham MA, USA #MT10040CV) with 10% FBS (Sigma-Aldrich, St. Louis, MO, USA, #F2442-500ML) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham MA, USA, #3MT30002CI) for 5 min at 37°C. Baseline fluorescence of Indo-1 AM loaded cells was acquired for 1 min LSR Fortessa X20 flow cytometer (BD Biosciences, San Jose, CA, USA). Subsequently DMSO (0.1%), CAD204520 1 µM, or thapsigargin 1 µM were added and measurement was resumed for a total of 10 min. Data analysis was performed using FlowJo V10 (Tree Star LLC, Ashland, OR, USA) analytical software. ER Ca2+ release and re-uptake was measured with the IonOptix system (IonOptix, Milton, MA, USA). Ca2+ signals were detected by epifluorescence after loading T-ALL cells (ALL/SIL, DND41) with Fluo-3-AM (10 µM; Invitrogen, Carlsbad, CA, USA) in PBS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) for 20 min, at RT. After removing the fluorophore, cells were washed with PBS for 30 min and then placed (1x106; 1ml volume) in a chamber mounted on the stage of an inverted microscope (Nikon-Eclipse TE2000-U, Nikon Instruments, Florence, Italy). The recording started with measurement of baseline fluorescence. Then, 1 µM CAD204520, 1 µM thapsigargin, or DMSO (0.1%) were manually 36 added with a pipette, and the recording was continued for up to 15 min (during the first 7 min: 5 seconds of recording followed by 5 seconds of rest; in the remaining 8 min: 5 seconds of recording followed by 30 seconds of rest). Excitation length was 480 nm, with emission collected at 535 nm. The following parameters were evaluated: (i) peak fluorescence normalized to baseline fluorescence (f/f0_peak), (ii) time at 50% of fluorescence signal decay, measured from the peak time (time_50%-f/f0), and normalized fluorescence computed at 3, 5, and 10 min from the peak time (f/f0_3min, f/f0_5min, and f/f0_10 min). Western Blot Protein lysates for western blotting were incubated with antibodies specific for g-secretasecleaved NOTCH1 (Val1744, #4147 or #2421 Cell Signaling, Beverly, MA, USA) or the Cterminus of NOTCH1 (#SC-6014 (C-20), Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cleaved form of Poly (ADP-ribose) polymerase was detected using an antibody specific for the cleaved peptide of PARP (#9541, Cell Signaling, Beverly, MA, USA). The expression of SERCA isoforms in ALL/SIL were detected using SERCA2 (#9580, Cell Signaling, Beverly, MA, USA) and SERCA3 (#sc-81759, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies while glucocorticoid receptor expression was detected using Glucocorticoid Receptor (D8H2) XP antibody (#3660; Cell Signaling, Beverly, MA, USA. Loading controls were performed with antibodies specific for b-Actin, (#BK3700S, Cell Signaling, Beverly, MA, USA), GAPDH (#137179, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or HSP90 (# sc-69703 (4F10)), Santa Cruz Biotechnology, Santa Cruz, CA, USA). Effects on the endoplasmic reticulum stress pathway (ER stress) we used the following antibodies: BiP, (#BK3177S), phospho-eIF2a (Ser51) (#9721S), eIF2a (#9722S) (Cell Signaling, Beverly, MA, USA). Blots were developed using species specific fluorescent antibodies obtained from 37 LI-COR (Biosciences, Lincoln, NE, USA) such as IRDye 680LT Goat anti-Mouse IgG (#92568020); IRDye 800CW goat anti-rabbit IgG (#925-32211); IRDye 680RD goat anti-rabbit IgG (#925-68071). Cell surface NOTCH1 was evaluated by staining non-permeabilized cells with monoclonal anti-human NOTCH1 antibody (#FAB5317P, R&D, Minneapolis, MN, USA). Indirect Immunofluorescence Microscopy DND41, REC-1 and ALL/SILL cells were resuspended in PBS, spotted on immunofluorescence slides (Thermo Fisher Scientific, Waltham, MA) by a cytospin centrifuge (CR2000, Small Prime Centrifuge, Centurion) fixed for 10 min in 4% paraformaldehyde (#28908, Thermo Fisher Scientific, Waltham MA, USA), permeabilized in 0.2% Triton X-100 for 5 min, and blocked in 5% bovine serum albumin for 1 hour. Then, the cells were incubated with primary antibodies against full length NOTCH1 (#SC-6014 (C-20) Santa Cruz Biotechnology, Santa Cruz, CA, USA or #ab44986 (A6) Abcam, Cambridge, United Kingdom), GOLGA1 (#SAB1409131, Sigma-Aldrich, St. Louis, MO, USA), and ATF6 (#37149, Abcam, Cambridge, United Kingdom). Alexa Fluor 488 (#A11029, Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 568 (#A11036, Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies and cells were incubated 1 hour at RT protected by the light. Nuclei were stained with DAPI (#D9542, Sigma-Aldrich, St. Louis, MO, USA). Coverslips were mounted with Prolong Gold Antifade reagent (#P36934, Thermo Fisher Scientific, Waltham MA, USA). Images were captured using a EVOS FL microscope (Thermo Fisher Scientific, Waltham MA, USA) and analyzed using ImageJ software (http://rsbweb.nih.gov/ij/). 38 Real-time RT-PCR Primers and probes for real-time RT-PCR were obtained from Applied Biosystems (Foster City, CA, USA) (RPL13A #Hs01926559_g1, MYC #Hs00153401_m1, DTX1 #Hs00269995_m1). Primers and probes used for glucocorticoid receptor quantitative RTPCR were obtained from Thermo Fisher Scientific (Waltham MA, USA) as following. For human GR alpha, forward primer: GAG-GAA-GTT-ATC-CTCTGC-CTC; reverse primer: TGT-AAG-CAC-CAC-CTTCCT-GTC-T; probe: 6FAM-TTC-CAA-CAG-TGA-GTCTGT-CAGCGC-A-QSY; for human GR beta, forward primer: GCT-GGA-TAA-TTA-GCA-TGG-GAT-G; reverse primer: AAT-TGC-TCC-CTG-CCT-CTG-A; probe: 6FAM-ATGAAG-GAA-AGCCAC-GCT-CCC-T-QSY. The data were analyzed using the DDCT method and plotted as percentage of transcript compared to vehicle. Values were considered statistically significant at P < 0.05. Whole Exome Sequencing DNA was extracted from about 10 x 106 ALL/SIL o ALL/SIL thapsigargin resistant using a Promega Maxwell™ kit as per the manufacturer’s protocol (Promega Corporation, Madison WI, USA, #AS1010). A total amount of 1.0 µg genomic DNA per sample was used as input material for the DNA library preparation. Sequencing libraries were generated using Agilent SureSelect Human all exon kit (Agilent Technologies, CA, USA) following manufacturer’s recommendations and index codes were added to each sample. Briefly, fragmentation was carried out by hydrodynamic shearing system (Covaris, Massachusetts, USA) to generate 180-280bp fragments. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and enzymes were removed. After adenylation of 3’ ends of DNA fragments, adapter oligonucleotides were ligated. DNA fragments with ligated 39 adapter molecules on both ends were selectively enriched in a PCR reaction. After PCR reaction, library hybridizes with liquid phase with biotin labeled probe, after which streptomycin-coated magnetic beads are used to capture the exons of genes. Captured libraries were enriched in a PCR reaction to add index tags to prepare for hybridization. Products were purified using AMPure XP system (Beckman Coulter, Beverly, USA) and quantified using the Agilent high sensitivity DNA assay on the Agilent Bioanalyzer 2100 system and sequenced with Hiseq PE 150 (Illuminaâ, San Diego, CA, USA). Paired-end clean reads were aligned to the reference genome (hg38) with Burrows-Wheeler aligner (B.W.A.). SAMtool was used to sort and index the original BAM files and Picard marked duplicates reads. Coverage and depth were calculated based on the final BAM files. If a read or reads pair were mapped to multiple positions, B.W.A. will choose the most likely position. If two or more likely position were present B.W.A. will choose one randomly. This multiple hit strategy has significant impact on SNP, INDEL and CNV detection, and variant calling accuracy. Following genomic variant detection, we performed annotation of variants with the tool ANNOVAR (132) in multiple aspects, including protein coding changes, affected genomic regions, allele frequency etc. Virus Production and Transduction of T-ALL Cell Lines 3 x 106 293T were plated in 10 cm plates and maintained in DMEM media (Life Technologies, Carlsbad, CA, USA, #11965118), 10% FBS (Sigma-Aldrich, St. Louis, MO, USA, #F2442-500ML), 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham MA, #3MT30002CI) and incubated at 37°C with 5% CO2, until sub confluent. Cells were transfected with 2 µg of pCMV-VSV-G envelope vector, Delta 8.9 packaging plasmid and pXPR-011-GFP, a vector expressing a green fluorescent protein (GFP), according to the 46 replacement with commercially available alternative heteroaromatic and heterocyclic systems, as well as pyridines substituted with small functional groups capable of picking up polar interactions. As a result, the piperidine analog 2-(2-piperidyl)-6-(trifluoromethoxy)-1Hindole (Figure 4BII) was identified as the minimum pharmacophore with improved SERCA ATPase potency, selectivity against Na+/K+-ATPase, and a reasonable ligand efficiency of 0.28. Options for diversity in substitution of indole C4-C7 were limited and C6-OCF3 was ‘‘locked’’ to continue exploration of more promising points of substitution. Furthermore, indole N1 had been extensively explored in a closely related chemical program with an overlapping pharmacophore, and for reasons of concern about target selectivity it was decided not to explore the indole N1 chemical space with the identified compound II. Conversely, compound II was explored in R1 of the indole system and R2 of the piperidine system (Figure S1B) for reasons detailed below. Compounds with substitution on piperidine N1 (R2) with R1 = H were subsequently produced and, among them, CAD204522, CAD307496, and CAD204521 (Figure 4B) showed various degrees of Ca2+-ATPase activity (Table S1). Interestingly, CAD204521 (Figure 4BIII; Table S1) was closely related to a previously reported potent fungal H+-ATPase inhibitor, Compound 7 (144). Similarly to Compound 7, CAD204521 showed an improved potency against Ca2+ ATPase but not a desired selectivity or drugability profile. Because indole C3 had the potential to provide Ca2+-ATPase selectivity, we decided to explore indole R1 with R2 = p-bromobenzyl (derivatives of CAD204521, Figure 4BIV). For example, CAD204519 was by far the most potent inhibitor of Ca2+-ATPase; however, with an unfavorable Na+/K+ Ca2+ selectivity. Nevertheless, substitutions with certain hydrophilic groups on the R2 on piperidine N1 (Figure 4B; Table S1, CAD306750, CAD306749, and CAD204520) increased the selectivity toward mammalian SERCA. 47 Notably, CAD204520 (Figure 4BV and S1C) preferentially inhibited the Ca2+-ATPase by reducing its ATP hydrolysis activity with an IC50 of 0.34 ± 0.03 µM as compared with Na+/K+- ATPase (IC50 = 8.30 ± 0.94 µM) and H+-ATPase (IC50 = 26.90 ± 2.98 µM) (Figure S1D; Table S1). Furthermore, CAD204520 displayed the overall most promising drug properties of the synthesized compounds with a calculated LogP of 4.4 and LogD7.4 of 2.2 (ACD/Labs 18.1.1). This compound was thus selected for further studies as a selective SERCA inhibitor. To assess the binding mode of CAD204520 to SERCA, we then crystallized it in complex with SERCA and determined the crystal structure at 3.4 Å resolution. The crystals were of the same space group as previously reported thapsigargin-bound SERCA (PDB: 2AGV), and the overall conformation of SERCA bound to CAD204520 is very similar to the thapsigargin-bound form. The CAD204520 ligand binds to a groove at the membrane interface of SERCA, between transmembrane helices M1, M2, M3, and M4 (Figures 4C and 4D), with two polar interactions to Asp59 on M1 (2.9 Å) and Asn101 on M2 (2.7 Å), and with several hydrophobic interactions involving Leu61, Val62, Ile307, Pro308, and Pro312 (Figure 4C). Interestingly, the CAD204520 binding groove is different from that of thapsigargin (Figure S1E), but similar to the binding of other SERCA inhibitors, such as CPA (6) (Figure S1F) and 2,5-di-t-butyl-1,4-benzohydroquinone (DBHQ) (28) (Figure S1G), and to that of the Compound 7 previously reported by Bublitz et al. (144) (Figure S1H). In fact, the indole system (core structure) of CAD204520 superposes very closely on the tetrahydrocarbazole core of Compound 7, including the central interaction of the indole N1 nitrogen with Asp59. The morpholinoethyl group, interacting with Asn101, occupies the same space as one of the two alternative positions found for the bromophenyl moiety of Compound 7 (Figure S1H). In contrast to Compound 7, however, there is no interaction with Asp245. Despite this similarity, CAD204520 does not induce the same overall SERCA conformation as Compound 7, but a 48 conformation almost identical to thapsigargin-inhibited SERCA. The thapsigargin-binding site lies adjacent to the CAD204520 site, separated by M3 (Figure S1I). Collectively, these data show that CAD204520 selectively binds SERCA in the same binding pocket as DBHQ, CPA, and Compound 7. This pocket has been identified as the pathway for Ca2+ ion entry into the pump from the cytosolic side of the membrane (29), and compound binding at this site locks SERCA in a Ca2+-free (so-called E2) conformation. CAD204520 Rescues T-ALL Cells from Thapsigargin Resistance SERCA can be inhibited by different small molecules, such as thapsigargin, DBHQ, 1,3dibromo-2,4,6-tris (methyl-isothio-uronium) benzene, and CPA. These compounds have specific binding sites in the ATPase protein and hence different inhibitory mechanisms (146). A first question is whether CAD204520 binding to SERCA mimics thapsigargin ATPase inhibitory kinetics or, rather, the two molecules act independently as predicted by structural data. To test our hypothesis, we took two different approaches. First, we generated a T-ALL cell line (ALL/SIL) resistant to thapsigargin (ALL/SIL R) by selecting cells growing under increasing concentration of this molecule. At approximately days 90, 120, and 150, T-ALL cells displayed 2-, 10-, and 27-fold increased IC50 values, respectively (Figure S2A). To rule out that this drug resistance was mediated by altered expression of the target, we demonstrated that naive and resistant cell lines showed similar levels of SERCA2 and SERCA3 proteins (Figure S2B). To evaluate for thapsigargin-induced gene mutations within the ATP2A1–3 genes, we performed whole-exome sequencing and limited our analysis to single-nucleotide exonic missense variation with a Phred-scaled quality score >30 (standard error = 1/1,000 = 0.1%; accuracy 99.9%) (Figure S2C). Previous work had demonstrated 49 that mutations occurring in the third stalk (M3) segment of SERCA determine the sensitivity of ATPase to thapsigargin (26, 147, 148). In particular, mutations in the M3 segment between Asp254 and Leu260 increase the thapsigargin concentrations required for inhibiting SERCA by more than three orders of magnitude (148, 149) (Figure S2D). Interestingly, in ALL/SIL R cells we identified, within the Asp254-Leu260 hotspot, a missense single-nucleotide polymorphism occurring in ATP2A2 exon 8 (c.G770T) caused a glycine257/valine mutation in the M3 helix (Figures 5A and S1I highlighted in red). No mutations occurred in ATP2A1 (Figure 5A, top panel), while missense mutations in ATP2A3 were present both in the naive and resistant lines, indicating a pre-existing mechanism of allelic variance (Figure 5A, bottom panel). Similarly, no acquired mutations were identified in SEC24A, a gene involved in ER-Golgi protein trafficking and previously identified as an essential mediator of thapsigargin-induced cell death in a genome-wide CRISPR/Cas9 screen in HAP1 cancer cells (150). According to our crystal structure, Gly257 faces a hydrophobic part of CAD204520 at a distance that could probably accommodate a valine residue without interfering with CAD204520 binding (Figure S1I). The introduction of the bulky valine side chain will, however, very likely limit the freedom of movement of the neighboring residue Phe256, which has to swing sideways to accommodate thapsigargin binding (Figures S1E and S1I), thus providing a potential explanation for the resistance effect. Next, we treated ALL/SIL naive and resistant cells at ALL/SIL IC50 (as shown in the following sections) concentrations and demonstrated that G257àV rescues ALL/SIL cells from thapsigargin-induced cytotoxicity while it does not interfere with CAD204520 effects (Figure 5B). Accordingly, because CAD204520 binds to SERCA in a pocket similar to that of CPA but distinct to that of thapsigargin, we anticipated that the combined inhibition might result in a synergistic effect with thapsigargin but not with cyclopiazonic acid. To avoid the 50 limitations and biases associated with any one algorithm used to study drug-drug interactions, we used comprehensive approaches, including the Loewe additivity model, the Chou and Talalay index, and the Bivariate Response to Additive Interacting Doses (BRAID) analysis. The Loewe additivity is a commonly used dose-effect-based model to quantify a zero-interactive state for the combination of two drugs (151). The Chou-Talalay method (152) for drug combination is based on the median-effect equation and provides a mechanism-independent method for quantitative determination, combination index (CI), of drug interactions. A CI ranging from 0.9 to 1.1 is considered additive, a CI < 0.9 indicates synergy, and a CI > 1.1 resistance. Finally, we used a response surface method, the BRAID model of combined action (153, 154). A κ BRAID index > 0 shows synergy between the compounds tested. Unlike most methods that reduce combination analysis to a simple decision between synergy, additivity, and antagonism, surface models use non-linear optimization to fit a response surface model to the effects of combined compounds. We tested CAD204520 and thapsigargin both individually and in combinations at the indicated concentrations for a total of 60 combinatorial points in T-ALL cell lines and in primary NOTCH1-mutated T-ALL samples. We found that simultaneous exposure to CAD204520 and thapsigargin for 72 h resulted in a robust synergistic inhibition of cell viability in T-ALL cells. Loewe, CI, and BRAID models established a synergistic effect at low-dose combinations and support the notion that CAD204520 binds at a site within SERCA that is distinct from the thapsigargin-binding sites (Figures 5C–5E). Consistent with our hypothesis, combined CAD204520 and CPA treatment did not demonstrate the same degree of synergistic activity (Figure S2E). Collectively, these data indicate that G257V mutation in the M3 helix of SERCA do not interfere with CAD204520 activity and that a greater anti-leukemia effect may be achieved 51 by the simultaneous binding of CAD204520 and thapsigargin to their respective sites in SERCA. CAD204520 Suppresses Leukemia Growth in NOTCH1-Mutated T-ALL and MCL We previously demonstrated that SERCA inhibitors decrease T-ALL growth both in vitro and in vivo (75). To validate CAD204520 as a potential modulator of Notch-dependent cancers we initially tested the effect of CAD204520 in a panel of T-ALL or MCL cell lines that contain activating mutations in the HD of NOTCH1 and/or deletions in the degradation domain (PEST) (Figure S3A). NOTCH1-mutated T-ALL (Figure 6A) (ALL/SIL, CTV-1, DND41, PF382, and RPMI-8402) or MCL cell lines suppressed by GSI (REC-1) (Figure 6B) (91) were more sensitive to CAD204520 as measured by inhibition of cell viability compared with NOTCH1 wild-type tumor cells (Figures S3B and S3C). Seventy-two hours of CAD204520 treatment triggered concentration-dependent apoptosis as determined by the increase of Annexin V/PI+ cells (Figure 6C) and the cleavage of PARP proteins (Figure 6D). An additional phenotypic consequence of NOTCH1 inhibition with GSI is that T-ALL cells undergo cell-cycle arrest (91). As shown in Figure 6E, CAD204520 induced a G0/G1 arrest preferentially in NOTCH1-mutated tumors (Figure S3D), and together with the data described above it supports the notion that CAD204520 inhibits lymphoid-derived cancer cells carrying clinically relevant NOTCH1 HD or PEST mutations. CAD204520 Suppresses Notch1 Signaling NOTCH receptors undergo several processing events, including a first cleavage by a furinlike convertase (S1) in the trans-Golgi network that generates full-length heterodimers (155, 156) ready to be conveyed to the plasma membrane (157). The correct folding of these 52 heterodimers requires Ca2+ that, in physiological condition, is tightly regulated across the ER storage by SERCA (68). To support the hypothesis that CAD204520-mediated SERCA inhibition impairs mutant NOTCH1 maturation, we evaluated the expression of NOTCH1 full-length and transmembrane portions of CAD204520-treated cells by western blotting. Lysates from TALL cell lines treated with 5 μM CAD204520 for 24 h were immunoblotted with an antibody specific for the cytoplasmic portion of NOTCH1 that recognizes both unprocessed NOTCH1 (FL-N1) (270 kDa) and the furin-processed transmembrane subunit (TM-N1) (110 kDa). CAD204520 reduced the levels of the furin-processed transmembrane NOTCH1 subunit, but not the unprocessed full-length NOTCH1 precursor, in multiple T-ALL cell lines (Figure 7A). As expected from our structural data, combined CAD204520 and thapsigargin treatment resulted in an enhanced reduction in ICN1 and TM-NOTCH1 levels (Figure S4A). In addition, we demonstrated that treatment of T-ALL with CAD204520 resulted in a concentration-dependent decrease in NOTCH1 expression on the cell surface by flow cytometry (Figure 7B). As expected, we did not observe this effect with a known GSI Notch inhibitor N-[N-(3,5-difluorophenacetyl)-1-alanyl]-(S)-phenylglycine (DAPT). Consistent with our hypothesis that CAD204520 affects NOTCH1 maturation rather than expression, NOTCH1 only decreases at the surface of the cells upon CAD204520 treatment but colocalizes at the ER-Golgi intermediate compartment as shown by immunofluorescence colocalization studies (Figures 7C and S4B–S4E). An immediate consequence of the decrement in NOTCH1 on the surface of the cells is the reduction of the catalytic activity of the g-secretase complex because of the lack of NOTCH1 substrate. Here, we would expect a reduction in the level of ICN1. Indeed, CAD204520 ultimately leads to loss of ICN1 (Figure 7D) and results in the suppression of NOTCH1 target genes MYC and DTX1 as measured 53 by RT-PCR (Figure 7E). Furthermore, testing CAD204520 in MCL NOTCH1-mutated cells yielded results comparable with the one described in T-ALL, suggesting a conserved mechanism across different NOTCH1-mutated cancers (Figure S4D-F). In summary, these data show that CAD204520 inhibits Notch1 maturation, demonstrating that SERCA inhibitors with a binding mode different from thapsigargin can efficiently suppress NOTCH1 maturation. CAD204520 Preferentially Inhibits NOTCH1-Mutated Cancers SERCA inhibitors increase the Notch therapeutic index by targeting clinically relevant NOTCH1 mutations in leukemia cells (75, 119, 158). In fact, leukemia cells carrying NOTCH1 alleles with HD mutations are more sensitive to SERCA inhibition than cells with wild-type NOTCH1 alleles (75, 119). To verify the hypothesis that CAD204520 preferentially targets mutant NOTCH1, we used two T-ALL cell lines carrying the same t(8; 14) (q24; q32)/TRAD@-MYC translocation but different Notch mutational status (Figures 8A and S5A). SKW-3/KE-37 harbors an isolated NOTCH1 mutation in the PEST domain, while MOLT16 is NOTCH1 wild type (91, 159). First, we determined that the mutant T-ALL cell line was more sensitive to CAD204520 growth inhibition as measured by an ATP-based cell viability assay (Figure S5B). To validate this observation, we established a flow cytometry-based competition assay. SKW-3/KE-37 were transduced with a GFP lentiviral expressing vector and co-cultured with MOLT16 T-ALL cells in a 1:1 ratio. Next, we treated SKW-3/KE-37-GFP and MOLT16 cocultured cells with increasing concentrations of CAD204520 and demonstrated that the mutated T-ALL cell line, SKW-3/KE-37-GFP, was more sensitive to growth suppression compared with wildtype MOLT16, as quantified by flow cytometric analysis of alive versus dead cells (Figure 54 8B). Analysis of caspase-3 and -7 activities indicates that the NOTCH1 mutational status sensitizes cells to CAD204520-mediated apoptotic cell death (Figure 8C). Consistent with the hypothesis that SKW-3/KE-37 relies on Notch signaling for growth and survival, we observed a decrement of NOTCH1 protein only in mutant T-ALL cells compared with wildtype cells (Figure 8D). To further support the preclinical development of CAD204520, we tested it (dose range = 0.6–8 μM) in a collection of T cell lymphoblasts isolated from T-ALL patients. As shown in Figure 8E, CAD204520 preferentially affects T-ALL viability compared with normal lymphocytes. Primary blasts, derived from a patient suffering from a NOTCH1-mutated TALL, exposed to 5 μM CAD204520, rapidly underwent apoptosis (Figure S5C). In addition, T-ALL primary cases for which we confirmed a NOTCH1 mutation (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) were more sensitive to CAD204520 compared with NOTCH1 wildtype B cell ALL (Figure 8F). Collectively, these results indicate that CAD204520 retains anti-tumor activity preferentially in cells carrying NOTCH1 alleles with HD or PEST mutations, holding great promise for CAD204520 future development against this indication. Consequences of Ca2+ Release upon CAD204520 Treatment A consequence of SERCA inhibition is the rise of intracellular Ca2+ followed by the depletion of Ca2+ stored in the ER. ER Ca2+ exhaustion triggers a number of secondary events, including the activation of the unfolded protein response (UPR) pathway (160), the activation of store-operated Ca2+ entry (161), and ultimately cell death (162). 55 To quantify the consequences of CAD204520 or thapsigargin treatment at the level of cytosolic Ca2+, we transferred ALL/SIL or DND41 in Ca2+-free media and loaded with Indo1 a ratiometric sensitive indicator fluorescent dye for measuring intracellular Ca2+. As shown in Figure 9A compared with DMSO, CAD204520 slightly increases cytosolic Ca2+. However, if compared with the thapsigargin effect, the extent of the increase appears modest with broad and flat peaks. In fact, thapsigargin causes a sharp rise in Ca2+ concentration at ~ 200 s upon drug injections. The next question is whether SERCA is still able to re-load Ca2+ from the cytosol inside the ER upon CAD204520 treatment. This hypothesis would explain why the increase of Ca2+ upon CAD204520 treatment is moderate and why thapsigargin triggers delayed on-target Ca2+ effects such as UPR activation and apoptosis (163). In this case we used a different approach and measured Ca2+ Fluo-3 AM epifluorescence using an IonOptix system. This approach is ideal to measure fluctuations of ER Ca2+. As reported in Figure 9B, the peak fluorescence (f/f0_peak) was similar in the three groups of cells, indicating that the different compounds did not modify the Ca2+ release from the ER. Conversely, the fluorescence signal decay was significantly prolonged in thapsigargin-treated cells in comparison with both CAD204520 and DMSO (time_50%-f/f0; p < 0.05). In accordance with this finding, the fluorescence computed at 3, 5, and 10 min from the peak time (f/f0_3 min, 5 min, and 10 min) or the area under the dose curve calculated within the same time frame (control versus CAD204520 Δmean = 0.4188; control versus thapsigargin Δmean = -2.658; CAD204520 versus thapsigargin Δmean = -3.077), was significantly higher only in the thapsigargin group (p < 0.05 and p < 0.0001, respectively), suggesting that CAD204520 did not delay the cytosolic Ca2+ reuptake. As previously mentioned, in addition to the effects on Ca2+ dynamics, thapsigargin and thapsigargin analogs activate the ER-related stress pathway of the UPR (164). To compare 62 DISCUSSION Although the prognosis of T-ALL has improved over the last two decades, the outcome of T-ALL patients with primary resistant or relapsed disease remains poor (173, 174). Therefore, current research goals are focused on the identification of targets to develop more effective and less-toxic anti-leukemic agents (100, 175-177). Several studies strongly support the development of Notch inhibitors for targeted therapy in hematological malignancies and solid tumors where Notch signaling is deregulated (178). For example, pan Notch pathway antagonism with GSIs reduces leukemia growth in mutant cancer cell lines and in mouse models (91, 179). Thus, modulators of Notch would be expected to have clinical efficacy particularly in T-ALL where recurrent NOTCH1 mutations are common and cancer dependency has been well established. However, prolonged suppression of the canonical Notch pathway in normal tissue may cause dose-limiting gastrointestinal toxicity (103) or increase the risk of skin cancers (180, 181), underscoring the need for new therapeutic modalities to preferentially suppress the oncogenic signal. In recent years, we have pursued this approach and demonstrated that selective inhibitors of SERCA, such as thapsigargin and CPA, uniquely among Notch inhibitors, preferentially affect mutated NOTCH1 proteins compared with the wild-type ones (75, 119). Thapsigargin, a plant-derived sesquiterpene-g-lactone, has been used extensively as a pharmacological tool to trigger Ca2+-dependent and UPR pathways in several disease models (182). Because the increase of cytosolic Ca2+ and sustained UPR activation (ER stress) are important mediators of apoptosis, SERCA inhibitors have been considered for cancer therapies (183). However, large-scale isolation from Thapsia or scalable synthesis of thapsigargin is complex, requiring a 5to 42-step process depending on the protocol used 63 (184-186). Similarly, the synthesis of thapsigargin-based derivatives presents significant challenges. In fact, thapsigargin itself possesses a polyoxygenated 5-7-5 tricyclic core linked to four diverse ester groups and eight stereogenic centers not suitable for structural modeling (184, 187). Structure-activity studies revealed that only few thapsigargin groups, for example, the ester bond at O(8), can be hydrolyzed to generate intermediate derivatives that can be used for conjugation with a peptide (188, 189) or with a cleavable ester linkage (119). In addition, modification of the thapsigargin ester acyl group, or of the lactone carbonyl, significantly reduces thapsigargin activity in cells or biochemical assays, preventing their broad applicability in cancer (190, 191). A further limitation is that native thapsigargin is not tractable as a therapeutic agent due to expected Ca2+ shifts that can be prevented, for example, by creating inactive pro-drugs activated in a hysto-specific manner (116, 192). This is the mode of action of mipsagargin, a thapsigargin derivative currently undergoing clinical trials for solid tumors (183). Our group has developed JQ-FT, a folate-thapsigargin derivative that leverages the dependency of leukemia cells on folate metabolism to direct the inhibitor into T-ALL cells (119). Another strategy is to exploit analogs that possess an enhanced selectivity toward SERCA isoforms preferentially expressed in cancer cells (115, 193), while keeping the activity of SERCA2a, the major cardiac isoform, unaffected (194-196) (Figure 3). An alternative is the development of small molecules that retain SERCA inhibitory capacities but have only transient effects on cytosolic Ca2+ shifts. This idea emerged from recent studies from the laboratory of Møller and colleagues that challenged the consensus idea that the elevation of cytosolic Ca2+ — rather than the depletion of ER Ca2+ — led to the cell death induced by thapsigargin and analogs (164, 197). Contrary to what is generally thought, the rapid rise of cytosolic Ca2+, as observed with thapsigargin, and its role in the short-term 64 side effect on cardiac contractility, is not required for apoptosis after SERCA inhibition. It is rather the ER Ca2+ depletion and sustained UPR activation that contributes to cell death (164). The effect of a given SERCA inhibitor on cytosolic and ER Ca2+ levels depends strongly on its molecular mechanism of interaction with the ATPase. For example, the thapsigargin derivative substituted with a 12-aminododecanoyl linker, Boc-8ADT, did not show measurable changes in Ca2+ levels even though it strongly inhibited SERCA ATPase activity (164) leading to apoptosis in LNCaP cells (198). This is probably due to the very slow binding kinetics of this compound leading to a slow net leakage of Ca2+ from the ER, which likely enables the maintenance of constant, stable cytosolic Ca2+ levels. Other possible causes for the lack of cytosolic Ca2+ peaks are a moderate decrease in SERCA’s Ca2+ affinity or a residual Ca2+ transport activity in the presence of the compound. It is tempting to speculate that other SERCA inhibitors that have advanced to clinical testing might have a similar mode of action. Curcumin, a small molecule derived from the turmeric spice that stabilizes SERCA in the E1 conformational state has been extensively tested in multiple cancer models and clinical trials (199) without causing major cardiac events. Cisplatin is a widely used platinumcontaining compound that, among other effects, inhibits SERCA and Na+/K+-ATPase simultaneously (200). Given the large number or rotating bonds in CAD204520, slow binding kinetics to SERCA, as with Boc-8ADT, can also be anticipated. From our structural data, the interaction of CAD204520 with SERCA involves only two polar contacts, and one single hydrophobic contact within a distance of 3 Å. Overall, the interaction looks surprisingly ‘‘loose’’, perhaps indicating a concentration-dependent competition with Ca2+ binding and transport rather than an irreversible inhibition. 65 Our data also show that CAD204520 binds to SERCA differently from thapsigargin: it occupies a pocket between the transmembrane helices M1, M2, M3, and M4 of SERCA, whereas thapsigargin binds between M3, M5, and M7 (59). This observation agrees with our finding that thapsigargin, but not CPA, co-treatment enhances CAD204520’s inhibitory effect, a feature that can be used for further medicinal chemistry optimization. In this regard, however, CAD204520 maintains the same thapsigargin ‘‘property’’ to preferentially alter mutated NOTCH1 trafficking. Remarkably, this ability has not yet been explored in two of the most recently synthesized putative SERCA inhibitors: the natural tricyclic clerodane diterpene casearin J (201) or ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2oxoethyl)-4H-chromene-3-carboxylate (CXL017) (202), both active in T-ALL cell lines. The next question is whether CAD204520 activity has limitations in vivo due to Ca2+ shifts. For example, mice exposed to a thapsigargin analog, L12-ADT, at 0.8 mg/kg, die within 8 h, putatively from cardiac toxicity (73). Cardiac SR Ca2+ ATPase (SERCA2a) plays a central role in myocardial contractility. SERCA2a actively transports Ca2+ into the SR and regulates cytosolic Ca2+ concentration, SR Ca2+ load, and thus the rate of contraction and relaxation of the heart (203). The amount of Ca2+ release from the SR, dictating the extent of cell shortening, is also a steep function of SR Ca2+ content (204). It follows that pharmacological inhibition of SERCA2a activity should reduce the amplitude of the transient calcium and the rate of SERCA-mediated Ca2+ removal, resulting in altered cardiomyocyte mechanics, as we observed in isolated unloaded ventricular myocytes exposed to CAD204520 or thapsigargin. However, the impairment of cellular contractile performance and Ca2+ dynamics was more pronounced after thapsigargin incubation compared with CAD204520 exposure (80%–90% reduction in functional performance versus 25%–30%, on average), 66 indicating that CAD204520 should have a better therapeutic window than thapsigargin in vivo. An important question is whether and to what extent the depressed cardiomyocyte function secondary to pharmacological inhibition of SERCA2 activity would translate into decreased cardiac function in vivo. Based on previous experience from our group in a rat model of induced cardiomyopathy (205), a 20%–30% decline in cellular mechanics ex vivo results in a comparable moderate hemodynamic impairment in the intact animal. In fact, while CAD204520 exerts an anti-leukemia effect in vivo it does not induce heart failure in the two different mouse models (BALB/c CD1 and IL2-NSG) used for this study. Although most NOTCH1 mutations are found in exons 26 and 27 coding for the HD region, mutations in the PEST domain are present in 20%–30% of tumors resulting in an increased Notch activation due to the prolonged stabilization of ICN1 (91). Activating mutations clustered in the PEST sequence have been described in CLL and in MCL, and several efforts are ongoing to target NOTCH1 in these diseases (92, 118). Our study demonstrates that CAD204520 is active in cell lines carrying a PEST mutation (SKW-3/KE-37). This result supports testing SERCA inhibitors in disease models with this recurrent abnormality, such as CLL (206) and MCL (92). To this end, we extended testing CAD204520 in the REC-1 MCL cell line, one of the few representative models of NOTCH1-dependent MCL (92) carrying a H2428Pfs*7 PEST mutation. We showed that REC-1 is sensitive to CAD204520 inhibition compared with NOTCH1 wild-type MCL lines. In REC-1, CAD204520 reduces Notch activation with a mechanism similar to the one observed in T-ALL. Because in MCL NOTCH1 mutations are associated with significantly shorter survival rates (92, 207), the development of Notch-targeted therapy may represent an effective strategy to tackle this aggressive disease. 67 Finally, since SERCA inhibitors display a favorable therapeutic index by targeting mutated NOTCH1 proteins, the development of new SERCA inhibitors, such as CAD204520 is a reasonable strategy for NOTCH1-mutated malignancies. For this reason, to anticipate the potential mechanism of resistance to SERCA inhibitors can improve the deep characterization of this class of molecule and speed up the translation into a clinical setting. To this purpose, we performed a small molecule screening on two T-ALL cell lines, respectively sensitive and resistant to the effect of thapsigargin, and identified glucocorticoids among the top classes with high activity in the resistant cell line. The modulation of SERCA activity due to the hotspot mutation in the thapsigargin binding site induced an upregulation of glucocorticoid receptor with the subsequent reversal of steroid resistance. Furthermore, glucocorticoids showed a synergistic activity with SERCA inhibitors especially in the resistant cell line, paving the way to a better understanding of SERCA activity and modulation in NOTCH1-mutated cancer. 68 CONCLUSIONS Modulation of intracellular Ca2+ homeostasis plays critical roles in key processes that regulate cellular survival, growth, differentiation, metabolism, and death in normal and cancer cells. Thus, it is not surprising that several anti-cancer agents suppress pro-survival and activate pro-apoptotic pathways through modulation of Ca2+ signaling-dependent mechanisms. This is, for example, the case for chemotherapeutics such as cytotoxic alkylating agents (208) or anti-metabolites that rely on a Ca2+ signaling component to induce cancer cell death (209). Similarly, natural compounds including alkaloids, flavonoids, diterpenoids, and polyphenolics have been extensively investigated for their ability to modulate intracellular Ca2+ concentration and participate in apoptotic signaling pathways. Among them SL, such as thapsigargin have been long regarded as target compounds for drug development. In fact, thapsigargin has a broad spectrum of growth suppressing activity in several tumor types including poorly dividing cells (210). However, we have demonstrated the SERCA inhibition may efficiently control the trafficking of NOTCH1 and that this blockade can be achieved without causing overt cardiac toxicities in preclinical leukemia models. Importantly the effects of SERCA suppression can be rescued by the overexpression of unprocessed NOTCH1 peptides such as ICN1 indicating that the anti-leukemia effect is on target for Notch inhibition rather than for more generic Ca2+ fluxes. An important standing question is why mutated NOTCH1 appears more sensitive to SERCA suppression compared to wild type isoform or other proteins more broadly. One hypothesis to explain NOTCH1 and SERCA functional dependency is by mechanisms of co-regulation. It has been previously shown that presenilin (PSEN) and SERCA co-localize in the ER (211). Since PSEN1 is a key regulator of NOTCH1 maturation and preferentially binds FL-N1 polypeptides processed through the ER, it is possible that NOTCH1-PSEN1-SERCA are 69 part of a co-functional protein complex. Interestingly, in a recent paper, treatment of T-ALL cell lines with the selective PSEN1 inhibitor MRK-560 inhibited mutant NOTCH1 processing and led to cell cycle arrest. MRK-560 treatment decreases leukemia burden and increased overall survival with no associated gut toxicity in T-ALL patient-derived xenografts in vivo suggesting that, similar to SERCA inhibition, disruption of PSEN1 may preferentially affect mutated proteins. The second hypothesis is a Ca2+ mediated one. In fact, Malecki and colleagues previously demonstrated that clinically relevant activating NOTCH1 HD mutations destabilize the NOTCH negative regulatory region and have deleterious effects on NOTCH1 folding and maturation. Because EGF and LNR repeats of NOTCH1 rely on Ca2+ for folding and activation, it may be possible that changes in ER Ca2+ may preferentially impair unstable NOTCH mutant proteins (212) compared to wild type providing a therapeutic window for SERCA inhibitors. Finally, a hypothesis not yet explored to explain FL-N1 accumulation at concentrations not sufficient to trigger the general mechanism of UPR is through a Ca2+ mediated transcriptional activation of inhibitors of furin-like proteases. This would explain for example why CAD204520 efficiently target cancers with isolated PEST deletions that would not be predicted to be unstable given a normal LNR and HD protein sequence. 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Immunofluorescence images of permeabilized ALL/SIL incubated with antiNotch1 (C20-red) and anti-Golgin (green) are shown. Co-localization is indicated by yellow signal. Scale bar, 100 µm magnification. D) Western immunoblot showing the expression of cleaved NOTCH1 (ICN1) in ALL/SIL, DND41, and RPMI-8402 cells treated at the indicated concentrations of CAD204520 for 24 h. HSP90 was used as a loading control. E) CAD204520 treatment for 24 h downregulates expression of NOTCH1 target genes in ALL/SIL, DND41, and RPMI-8402 T-ALL cells as assessed by qRT-PCR. Error bars indicate the mean ± SD of four replicates. Data were analyzed using the ΔΔCT method and plotted as a percentage relative to the control gene RPL13A. Statistical significance (***p ≤ 0.001, ****p ≤ 0.0001) was determined by one-way ANOVA using Bonferroni’s correction for multiple comparison testing. GSI (DAPT) was used as a positive control. Figure 8. NOTCH1 Mutation Sensitizes T-ALL Cells to CAD204520 Inhibition 95 A) Interphase and metaphase FISH, with the LSI MYC probe, show split signals between der(8) (red signal) and der(14) (green signal), in the MOLT-16 (left) and SKW-3/KE-37 (right) cell lines. (b) SKW-3/KE-37 has two der(8). B) Left: cell-based competition assay. SKW-3/KE-37 and MOLT16 were transduced with a GFP-containing vector or an empty control vector, respectively, and co-cultured at a 1:1 ratio. Right: normalized effect of CAD204520 on cellular viability in co-cultured SKW-3/KE37-GFP and MOLT16 cells treated for 72 h. Error bars denote the mean ± SD of two replicates for vehicle-treated (DMSO) cells and for CAD204520-treated cells. Statistical significance (*p ≤ 0.05) was determined by one-way ANOVA using Bonferroni’s correction for multiple comparison testing. C) Caspase-3/-7 luminescence fold induction in SKW-3/KE-37 and MOLT16 cells. Error bars denote the mean ± SD of six replicates for vehicle-treated (DMSO) cells and for CAD204520treated cells. Statistical significance (***p ≤ 0.001) was determined by one-way ANOVA using Bonferroni’s correction for multiple comparison testing. D) Effect of CAD204520 treatment for 24 h on NOTCH1 (N1) processing and activation in SKW-3/KE-37 and MOLT16 cell lines. The immunoblot was stained with an antibody against the C terminus of NOTCH1 that recognizes the furin-processed NOTCH1 TM and the unprocessed NOTCH1 precursor (FL). HSP90 was used as a loading control. E) Effect of the CAD204520 in primary T-ALL cells (n = 9) or isolated lymphocytes (n = 6). The whisker plot represents the effect of small molecules on cellular viability calculated using the area under the curve (AUC) model of log-transformed dose-responses data using GraphPad v.7. The line in the whisker diagram represents the AUC median. The upper edge (hinge) indicates the 75th percentile of the dataset, and the lower hinge the 25th percentile. 96 The ends of the vertical line show the minimum and the maximum data values. Statistical significance (***p ≤ 0.001) was determined by a non-parametric t test (Mann-Whitney). F) Normalized effect of the CAD204520 in primary NOTCH1-mutated T-ALL cells (n = 2) or primary B-ALL cells (n = 2) on cellular viability. Error bars denote the mean ± SD of four replicates. Statistical significance comparing each T-ALL versus B-ALL case to each dose (***p ≤ 0.001, ****p ≤ 0.0001) was determined by a non-parametric t test (Mann-Whitney). Figure 9. Effects of CAD204520 on Ca2+ and UPR Activation A) Indo-1 AM fluorescence traces of T-ALL cells loaded with 5 μM of Indo-1 AM and treated with DMSO, CAD204520 1 μM, or thapsigargin 1 μM. Baseline and post-treatment fluorescence is indicated by a black arrow. Cells were acquired for a minimum of 10 min on an LSR Fortessa X20 flow cytometer. B) Time course of ER calcium release and reuptake traces recorded in DMSO, CAD204520, and thapsigargin T-ALL-treated cells. Each trace is representative of five (DMSO) or four (CAD204520 and thapsigargin) independent experiments. In green the area under the curve (AUC). Values are reported as mean ± SEM; f/f0_peak, peak fluorescence normalized to baseline fluorescence; time_50%-f/f0, time at 50% of fluorescence signal decay measured from the peak time; f/f0_3min, 5min, and 10 min, fluorescence computed at 3, 5, and 10 min from the peak time. *p < 0.05 versus DMSO; #p < 0.05 versus 1 μM CAD204520. Statistical significance was determined by a Kruskal-Wallis test and differences among groups were determined by a Mann-Whitney non-parametric t test. C) Effect of CAD204520 and thapsigargin treatment for 24 h in ALL/SIL and DND41 cell lines. The blot was stained with an antibody against the C terminus of NOTCH1 that 97 recognizes the furin-processed NOTCH1 TM and the unprocessed NOTCH1 precursor (FL), an antibody that recognizes the cleaved NOTCH1 (ICN1), P-eIF2α, total eIF2α, BiP, and HSP90 used as a loading control. D) Effect of CAD204520 and thapsigargin treatment for 24 h on ATF6 in ALL/SIL cell line. Immunofluorescence of permeabilized ALL/SIL cells stained with ATF6 (green) is shown. Cell nuclei were stained with DAPI (blue). Scale bar, 100 mm. E) Effects of CAD204520 (left) and thapsigargin (right) on cell viability after 72 h of treatments in HL-1 and ALL/SIL cell lines. Error bars denote ± SD of a minimum of two replicates. (F) Effect of CAD204520 and thapsigargin treatment for 24 h in HL-1 cell lines. The blot was stained with BiP. β-Actin was used as a loading control. Figure 10. Effects of CAD204520 on Preclinical Model of T-ALL A–C) Left panels: effect of CAD204520 treatment on rat cardiomyocyte mechanics. Single experiments are represented by two dots interconnected by a solid line. Specifically, the line between the dots connects the quantification of maximal rate of shortening (A) (-dl/dtmax), maximal rate of re-lengthening (B) (+dl/dtmax), and fraction of shortening (C) (FS%), before and after the CAD204520 (5 μM) or thapsigargin (200 nM) treatment compared with control (Control). (A–C) Right panels: mean percentage effect of CAD204520 (CAD2045202h) and thapsigargin (Thapsigargin200nM) on the same cardiac functions. Graph bars: mean ± SD of the six CAD204520-treated cardiomyocyte groups and mean ± SD of the two thapsigargintreated cardiomyocyte groups. Statistical significance comparing CAD204520-treated cells 98 versus thapsigargin-treated cells (**p ≤ 0.001, *p ≤ 0.05) was determined by a nonparametric t test (Mann-Whitney). D) Effect of daily 30 mg/kg administration of CAD204520 on body weight. Error bars denote the mean ± SD of six replicates (three male and three female mice). Statistical significance (n.s.) was determined by a two-way ANOVA analysis. E) Effect of CAD204520 on T-ALL leukemia burden in an SKW-3/KE-37-xenografted murine model. Anti-leukemic activity of CAD204520 assessed by measuring hCD45+ cells after 4 days of CAD204520 treatment (45 mg/kg/OS BID) or vehicle (Tween 80 0.5%, w/v, and hydroxypropyl-methylcellulose [HPMC] 1.0%, w/v). Representative dot plot showing the effect of CAD204520 on T-ALL growth in an SKW-3/KE-37 murine model. A minimum of 20,000 events was collected for each condition. F) Immunohistochemical analysis of the spleen in an SKW-3/KE-37-xenografted murine model treated with CAD204520 45 mg/kg or vehicle for 4 days. The spleens of all mice were examined; representative results for one control animal and one CAD204520-treated animal are shown. Formalin-fixed, paraffin-embedded tissue sections were stained with hCD45. Scale bars, 20 μm. G) Representative images of hematoxylin/eosin-stained sections of the left ventricle from SKW-3/KE-37 xenograft treated with CAD204520 45 mg/kg or vehicle. CAD204520 treatment did not affect the gross structural components of the myocardium or induce focal areas of damage. Well-aligned myofibers in the absence of myocytolytic necrosis or interstitial inflammatory infiltrates are shown at higher magnification (inset). Scale bars, 0.2 mm (low magnification) and 0.05 mm (high magnification; insets). 99 H) Representative images of hematoxylin/eosin-stained histological sections of the small intestines from SKW-3/KE-37 xenograft treated with CAD204520 45 mg/kg or vehicle. Compared with controls, intestinal villi and crypts appear to be well preserved in CAD204520-treated animals. At higher magnification (inset), no morphological changes in goblet cells and enterocytes were observed in CAD204520-treated mice. Scale bars, 0.2 mm (low magnification) and 0.05 mm (high magnification; insets). I) Effect of CAD204520 on cell blood count WBC, hemoglobin, and platelets in an SKW3/KE-37 murine model after 4 days of CAD204520 treatment (45 mg/kg/OS BID) or vehicle (Tween 80 0.5%, w/v, and HPMC 1.0%, w/v). Error bars denote the mean ± SD of eight CAD204520-treated animals or the mean ± SD of eight replicates vehicle-treated mice. Statistical significance for treated versus vehicle (n.s.) was determined by non-parametric t test (Mann-Whitney). Figure 11. Resistance to SERCA inhibitors sensitize T-ALL cells to glucocorticoids A) Small molecule viability screen results of EU-OPENSCREEN (European Chemical Biology Library) in ALL/SIL (blue lines) and ALL/SIL R (red lines) cell lines. All molecules were tested at 100 nM concentration. Radar plot shows the effect of individual drugs. From the left: small molecules active (viability < 50%) on either ALL/SIL R, both cell lines or ALL/SIL only. B) Radar plots reporting drug screening viability results of compounds active on NR3C receptors in ALL/SIL (blue lines) and ALL/SIL R (red lines). All drugs were tested at 100 nM. Each subgroup of NR3C receptor is indicated with a different color. 100 C) Left: Western blotting showing expression of Notch1 pathway and total glucocorticoids receptor (GR) in T-ALL cell lines. Right: Real-time PCR results comparing the fold change in gene expression of glucocorticoids receptor isoforms in ALL/SIL and ALL/SIL R cell lines in basal conditions. The y axis represents the fold change between each condition and βactin expression as an internal control. Error bar denotes the mean ± SD of a minimum of three replicates. Statistical significance among groups (****p < 0.0001) was determined by one-way ANOVA. D) Effects of glucocorticoids on cell viability after 72 hours of treatments in T-ALL cell lines. Error bars denote ± SD of 2 replicates. E) Effects of glucocorticoids on cell viability after 72 hours of treatments in T-ALL cell lines in basal conditions and after a rescue with ALL/SIL R medium. Error bars denote ± SD of 2 replicates. Black: rescue conditions; pink: basal conditions. F) Effects of glucocorticoids on cell viability in ALL/SIL and ALL/SIL R T-ALL cell lines with the following conditions: after 72 hours of treatments, after 72 hours of treatments in combination with thapsigargin 10 nM; after a pre-treatment with RU486 1 μM and thapsigargin 10 nM for 24 hours and a following treatment with glucocorticoids for 72 hours. Error bars denote ± SD of 2 replicates. G) Combination index (left) and surface plots analysis (right) of ALL/SIL and ALL/SIL R TALL cell lines treated with vehicle, CAD204520, fluticasone, or CAD204520 plus fluticasone. Each point means an independent measurement representative of two biological replicates. Plots were generated using Combenefit script by MATLAB R2021, which represents the Loewe (dose-effect based approach) analysis. A color scale bar represents the level of drugs antagonism or synergism. 101 Figure S1 (Related to Figure 4 and Table S1): Synthesis Route, Activity and Binding Mode of CAD204520. A) Chemical structure of the initial hit compound: 2-(2-pyridyl)-6-(trifluoromethoxy)-1Hindole. B) Schematic representation for medicinal chemistry optimization. R1 and R2 substitutions are indicated. C) Synthesis route of CAD204520 (4-[2-[2-[3-propyl-6-(trifluoromethoxy)-1H-indol-2-yl]-1piperidyl]ethyl]morpholine). Synthetic route (a) to (e) is depicted and described in the methods section. D) Determination of the protein ATP hydrolysis activity in the presence of compound CAD204520 at pH 7. The figure displays ATPase activity determined by measuring the amount of liberated phosphate from ATP hydrolysis. Data is presented as a fitted curve which has been normalized to the maximal enzyme activity with subtraction of background signal from spontaneous hydrolysis of ATP. Error bars denote the mean ± SD (standard deviation) of 3 replicates. Statistical significance (**P ≤ 0.01; ***P ≤ 0.001) was determined by two-way ANOVA using Bonferroni’s correction for multiple comparison testing. E) Binding sites of CAD204520 and thapsigargin. Superposition of the SERCA-CAD204520 complex with SERCA-thapsigargin (PDB ID: 2AGV). The binding sites are both in the transmembrane region, separated by transmembrane helix M3. Thapsigargin is shown as cyan surface representation. 102 F) Superposition of SERCA-CAD204520 with SERCA-CPA (PDB ID 3FGO), viewed roughly along the membrane normal. CAD204520 and CPA are shown as orange and green sticks, respectively. G) Superposition of SERCA-CAD204520 with SERCA-DBHQ (PDB ID 2AGV) viewed roughly along the membrane normal. CAD204520 and DBHQ are shown as orange and light blue, respectively. H) Superposition of SERCA-CAD204520 with SERCA-Cpd7 (PDB ID 5NCQ), viewed along the membrane plane. CAD204520 and Cpd7 are shown as orange and magenta sticks, respectively. I) Binding sites of CAD204520 and thapsigargin, as seen roughly along the membrane normal. Superposition of SERCA bound to CAD204520 (light blue cartoon and orange sticks, respectively) with SERCA bound to thapsigargin (grey cartoon and sticks, respectively). Glycine257, which is mutated to valine in the thapsigargin resistant mutant, is indicated by a red sphere. In the SERCA-thapsigargin complex, Phe256 has undergone a displacement that is likely to be impaired by a valine residue in position 257. Figure S2 (Related to Figure 5): Identification of a thapsigargin-resistant T-ALL cell line. A) Effects of Thapsigargin (left) and CAD204520 (right) on cell viability after 72 hours of treatments in ALL/SIL and ALL/SIL thapsigargin-resistant cell lines. Error bars denote ± SD of a minimum of 2 replicates. B) Western blot showing the expression of SERCA2 and SERCA3 in naïve and resistant ALL/SIL. β-actin was used as a loading control. 103 C) Phred-scale analysis of exonic single nucleotide variation (SNV) occurring in the ALL/SIL thapsigargin resistant cell line. Inset shows number (N.) of variation (SNV) occurring per chromosome. D) Thapsigargin resistance mutation hot spot region on helix M3. Thapsigargin and SERCA residues 254-260 are shown in stick representation and colored cyan and red, respectively. E) Surface plots analysis of ALL/SIL, DND41 and RPMI-8402 T-ALL cell lines and a primary NOTCH1-mutated T-ALL sample treated vehicle, CAD204520, cyclopiazonic acid, or CAD204520 plus cyclopiazonic acid. Each point represents an independent measurement representative of three biological replicates. Plots were generated using Combenefit script by MATLAB R2018 and represent the Loewe (dose-effect based approach) analysis. A color scale bar represents level of drug antagonism or synergism. Figure S3 (Related to Figure 6): Effect of CAD204520 on NOTCH1 mutated and wild type T-ALL and MCL cell lines. A) Table representing NOTCH1 mutational status in T-ALL and MCL lines. B) Scatter dot plot representing IC50 [μM] of CAD204520 in NOTCH1 mutated (n = 5) or NOTCH1 WT (n = 3) T-ALL or in NOTCH1 mutated (n = 1) or NOTCH1 WT (n = 3) MCL (shown in C) cell line. Statistical significance (*P ≤ 0.05) was determined by a non-parametric t-test (Mann-Whitney). D) Effect of CAD204520 treatments on cycling MAVER-1 and MINO cells. Percentage of DNA content following four days of treatment with the indicated concentrations of CAD204520 on each cell cycle phase is indicated. A minimum of 20,000 events was collected for each condition. MOLT16 cell line t(8;14)(q24;q11) SKW-3/KE-37 cell line t(8;14)(q24;q11) CAD240520 0.0 0.5 1.0 1.5 Fraction of Cell Alive 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 Increase 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 Fraction of Cell Alive Area Under the Curve *** 0 50 100 150 Lymphocyte T-ALL FE Figure 8 -dl/dtmax (µm/s) % of variation Max Rate of Shortening (-dl/dtmax(µm/s)) Max Rate of Re-Lenghtening (-dl/dtmax(µm/s)) Fraction of Shortening (FS(%)) 0 20 40 60 80 100 ** A 0 20 40 60 80 100 +dl/dtmax (µm/s) % of variation ** B Control CAD204520 5 µ M 0 20 40 60 80 100 FS % of variation CAD204520 5 µM Thapsigargin 0.2 µM * Control Thapsigargin 0.2 µ M C Days 0 4 8 12 16 20 0 10 20 30 Vehicle CAD204520 30 mg/Kg Body Weight (gr) D E CD45-PE-A subset 5.64% 50K 100K 150K CAD204520 45 mg/Kg Vehicle CD45-PE-A subset 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 Figure 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