ASTX660, a Novel Non-Peptidomimetic Antagonist of Ciap1/2 and XIAP, Potently Induces Tnfa-Dependent Apoptosis in Cancer Cell Lines and Inhibits Tumor Growth George A

Published OnlineFirst April 25, 2018; DOI: 10.1158/1535-7163.MCT-17-0848

Small Molecule Therapeutics Molecular Cancer Therapeutics ASTX660, a Novel Non-peptidomimetic Antagonist of cIAP1/2 and XIAP, Potently Induces TNFa-Dependent Apoptosis in Cancer Cell Lines and Inhibits Tumor Growth George A. Ward, Edward J. Lewis, Jong Sook Ahn, Christopher N. Johnson, John F. Lyons, Vanessa Martins, Joanne M. Munck, Sharna J. Rich, Tomoko Smyth, Neil T. Thompson, Pamela A. Williams, Nicola E. Wilsher, Nicola G. Wallis, and Gianni Chessari

Abstract

Because of their roles in the evasion of apoptosis, inhibitor of antagonism of XIAP was demonstrated by measuring its dis- apoptosis proteins (IAP) are considered attractive targets for placement from caspase-9 or SMAC. Compound-induced pro- anticancer therapy. Antagonists of these proteins have the teasomal degradation of cIAP1 and 2, resulting in downstream potential to switch prosurvival signaling pathways in cancer effects of NIK stabilization and activation of noncanonical cells toward cell death. Various SMAC-peptidomimetics with NF-kB signaling, demonstrated cIAP1/2 antagonism. Treatment inherent cIAP selectivity have been tested clinically and dem- withASTX660ledtoTNFa-dependent induction of apoptosis onstrated minimal single-agent efficacy. ASTX660 is a potent, in various cancer cell lines in vitro, whereas dosing in mice non-peptidomimetic antagonist of cIAP1/2 and XIAP, discov- bearing breast and melanoma tumor xenografts inhibited ered using fragment-based drug design. The antagonism of tumor growth. ASTX660 is currently being tested in a phase XIAP and cIAP1 by ASTX660 was demonstrated on purified I–II clinical trial (NCT02503423), and we propose that its proteins, cells, and in vivo in xenograft models. The compound antagonism of cIAP1/2 and XIAP may offer improved efficacy binds to the isolated BIR3 domains of both XIAP and cIAP1 over first-generation antagonists that are more cIAP1/2 selective. with nanomolar potencies. In cells and xenograft tissue, direct Mol Cancer Ther; 17(7); 1–11. 2018 AACR.

Introduction members of the family, such as cIAP and XIAP, also possess RING (Really Interesting New Gene) zinc ﬁnger domains with E3 Evasion of apoptosis is one of the hallmarks of cancer (1) and ubiquitin ligase activity (6, 7). The antiapoptotic activity of XIAP can be achieved by overexpression of antiapoptotic proteins. is mediated by its direct binding to and inactivation of caspases 3, Inhibitor of apoptosis proteins (IAP), such as cellular IAP (cIAP) 7, and 9 via its BIR domains (8). IAP antagonists such as the 1 and 2 and X-linked IAP (XIAP), are key regulators of antiapop- endogenous second mitochondria-derived activator of caspases totic and prosurvival signaling pathways; XIAP directly inhibits (SMAC), which is released from mitochondria on induction of caspases, whereas cIAPs prevent the formation of proapoptotic apoptosis, bind to the BIR domains of IAPs and can disrupt signaling complexes. This leads to suppression of apoptosis interactions such as those between XIAP and caspase-9 (9). On through both the extrinsic and intrinsic apoptosis pathways binding to other IAPs (cIAP1 and cIAP2), SMAC induces a (2–4). Their deregulation, through ampliﬁcation, overexpression, conformational change, which activates their E3 ligase func- or loss of endogenous antagonists, occurs in various cancers and is tion, leading to rapid autoubiquitination and proteasomal associated with tumor growth and poor prognosis, making them degradation (10). attractive targets for anticancer therapy (5). In response to TNFa, cIAPs ubiquitinate RIP1, promoting the The IAPs are characterized by their baculovirus IAP repeat (BIR) formation of complexes (e.g., complex I), which activate survival domains, which mediate protein:protein interactions; some signaling through the canonical NF-kB pathway. Simultaneously, formation of the death-inducing signaling complex (DISC), which drives apoptosis, is prevented. Antagonism and subsequent Astex Pharmaceuticals, Cambridge, United Kingdom. degradation of the cIAP1/2 leads to the stabilization of NIK (NF- Note: Supplementary data for this article are available at Molecular Cancer kB-inducing kinase), which activates the noncanonical NF-kB Therapeutics Online (http://mct.aacrjournals.org/). pathway, resulting in the production of multiple cytokines includ- Corresponding Author: George A. Ward, Astex Pharmaceuticals, 430 Cam- ing TNFa. Removal of the cIAP1/2 also allows the DISC to form, bridge Science Park, Milton Road, Cambridge, CB4 0QA, United Kingdom. leading overall to a switch in TNFa signaling from prosurvival to Phone: 44-122-322-6200; Fax: 44-122-322-6201; E-mail: proapoptotic (11–15). This loss of cIAP1/2 combined with release [email protected] of the XIAP-mediated block on caspases, which is essential for full doi: 10.1158/1535-7163.MCT-17-0848 activation of apoptosis, leads to a sustained proapoptotic effect in 2018 American Association for Cancer Research. the presence of TNFa via the extrinsic apoptosis pathway. Tumors

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with sufficient levels of TNFa in their environment may, therefore, amide; Peptide Synthetics Ltd) by fluorescence polarization be particularly susceptible to IAP antagonism (16). In addition, on a Pherastar plate reader (BMG Labtech). IC50 curves were the antagonism of XIAP-mediated caspase inhibition promotes generated using GraphPad Prism version 6 and fitted using the apoptosis induced by stimulation of the intrinsic apoptosis path- four parameter logistic curve fit. way by agents such as chemotherapeutics or DNA-damaging agents (17). This suggests that cIAP1/cIAP2/XIAP antagonists can Cell lines be used to promote apoptosis through both the extrinsic and The human cell lines MDA-MB-231 and HEK293 were pur- intrinsic pathways. chased from the European Collection of Cell Cultures (ECACC); IAPs have been therapeutically targeted by antisense oligonu- human melanoma cell lines, A375 and SK-MEL-28, were pur- cleotides and antagonist small molecules (4). AEG35156, an chased from ATCC; and the diffuse large B-cell lymphoma cell antisense oligonucleotide targeted to XIAP, showed some evi- line, WSU-DLCL2, was purchased from DSMZ. All were grown in dence of clinical activity (18) and sensitized cancer cells to DMEM medium supplemented with 10% FBS and maintained at chemotherapeutic agents and TRAIL receptor agonists in preclin- 37C in an atmosphere of 5% CO except WSU-DLCL2 cells, fi 2 ical models (3). The rst generation of SMAC-mimetic IAP which were grown as above except in RPMI medium supplemen- antagonists to enter the clinic, all containing alanine moieties ted with 10% FBS. All cell culture reagents were purchased from and inherently cIAP-selective, have shown some activity in pre- Invitrogen unless stated otherwise. These cells lines were not – fi clinical models (19 22), but thus far limited single-agent ef cacy passaged for more than 6 months (or 30 passages) after authen- – in clinical trials (3, 4, 23 26). tication by the cell bank (short tandem repeat PCR) and were fi We have previously reported the identi cation of lead com- routinely screened for mycoplasma (MycoAlert, LONZA). Mela- pounds with activity against cIAP1 and XIAP by fragment-based noma cell lines screened at ChemPartner were purchased from screening and structure-based drug design (27, 28). Here, we ATCC, except COLO679 (ECACC), GAK (Japanese Collection of describe the discovery and characterization of ASTX660, an antag- Research Bioresources), MMAC-SF (Riken Cell Bank), and normal onist of cIAP1/2 and XIAP, which is currently being tested in a human dermal fibroblasts (NHDF; LONZA), and were mycoplas- – phase I II clinical trial (NCT02503423). We hypothesize that ma screened, short tandem repeat PCR verified, and not used fi such IAP antagonism may lead to improved ef cacy as a result of beyond 10 passages. the more effective activation of apoptosis provided by blocking cIAP1/2 while releasing the XIAP block on caspases. Western blots Cell lysate samples were resolved by SDS-PAGE and immuno- Materials and Methods blotted as described previously (27) with antibodies specific Materials for XIAP (AF8221), cIAP1 (AF8181), and cIAP2 (AF8171) from ASTX660 as the hydrochloride salt was synthesized using a R&D Systems, and the following antibodies from Cell Signaling chemical procedure similar to that used for AT-IAP (27). The Technology: cleaved caspase-3 (#9664), cleaved PARP (#9541), key step involved the coupling reaction between methyl-5-((R)-3- cleaved caspase-9 (#9505), phospho-p65 (S536; #3033), methyl-morpholin-4-ylmethyl)-piperazine-1-carboxylic acid tert- phospho-IkBa (Ser32; #2859), total IkBa (#4814), NF-kB1 butyl ester and 2-chloro-1-{6-[(4-fluorophenyl)methyl]-5- (p105/p50; #3035), NF-kB2 (p100/p52; #4882), SMAC (hydroxymethyl)-3,3-dimethyl-1H,2H,3H-pyrrolo[3,2-b]pyridin- (#2954), NIK (4994), and b-actin (#8457). 1-yl}ethan-1-one (see Supplementary Materials and Methods); purity was determined as greater than 95% by high-performance Immunoprecipitation with anti-XIAP liquid chromatography. All other reagents were purchased from Equivalent amounts of cell lysate were incubated overnight at Sigma unless otherwise stated. BV-6 [(2S)-2-{[(2S)-1-[(2S)-2-cyclo- 4C with protein A/G magnetic beads (Pierce) coated with anti- hexyl-2-[(2S)-2-(methylamino)propanamido]acetyl]pyrrolidin- XIAP polyclonal antibody (R&D Systems). The beads were 2-yl]formamido}-N-{6-[(2S)-2-{[(2S)-1-[(2S)-2-cyclohexyl-2- washed and boiled in SDS sample buffer containing DTT, before [(2S)-2-(methylamino)propanamido]acetyl]pyrrolidin-2-yl]for- analysis of the eluted proteins by Western blotting. Western blots mamido}-3,3-diphenylpropanamido]hexyl}-3,3-diphenylpro- of the same lysate before immunoprecipitation were used for panamide] (14) was purchased from Selleckchem. comparison. Antagonism of XIAP by ASTX660 was monitored by Western blotting for levels of SMAC immunoprecipitated by Protein production and crystallography XIAP. A XIAP-BIR3 construct (amino acids 250-354) was expressed in E. coli, purified by affinity and size exclusion column chromatog- Meso Scale discovery assays raphy, and crystallized at approximately 10 mg/mL as described An engineered HEK293 cell line (clonal isolate from stable previously (27). Crystals were soaked with 5 mmol/L ASTX660 transfection with full-length FLAG-tagged XIAP expression in 5% DMSO overnight at room temperature prior to data construct (RC207627) and a full-length untagged caspase-9 collection. The crystals had cell dimensions of approximately construct (SC119362; Origene) were used to detect XIAP 70 Å 70 Å 105 Å and belong to space group P4122. The binding to caspase-9. Cells were incubated with compound diffraction observed ranged from 1.7 to 3.0 Å. for 2 hours, lysed, and lysates added to streptavidin Meso Scale Discovery (MSD) plates coated with biotinylated anti-XIAP Binding assays polyclonal antibody (R&D Systems), before washing and Interaction between ASTX660 and the BIR3 domains of XIAP or probing with anti–caspase-9 or anti-XIAP antibody (Cell cIAP1 was determined by measuring the displacement of a fluo- Signaling Technology), followed by the appropriate secondary rescent peptide tracer derived from SMAC (AbuRPFK(5&6FAM)- detection antibody.

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ASTX660 is an Antagonist of cIAP1/2 and XIAP

An MSD plate-based assay was used to quantify levels of cIAP1 ately snap frozen in liquid nitrogen before being stored at 80C in MDA-MB-231 after 2-hour ASTX660 treatment. Cells were prior to analysis. incubated with compound for 2 hours, washed, and lysed. Lysates MDA-MB-231 xenografts were prepared by subcutaneously were applied to MSD plates as described previously (27). injecting 5 106 cells, suspended in 100 mL of serum-free medium, into the right hind flank of male SCID (BALB/ scid Live cell imaging cJHan Hsd-Prkdc ) mice. A375 xenografts were prepared by Cells were imaged in real time using the IncuCyte ZOOM live subcutaneously injecting 5 106 cells, suspended in 100 mLof cell imager (Essen BioScience). Cells were incubated with com- a 1:1 mixture of serum-free medium and Matrigel (approximately pound in 0.1% (v/v) DMSO, with or without neutralizing anti- 10 mg/mL, Corning), into the right hind flank of male nude mice nu TNFa antibody (R&D Systems) for 5 days, and live images were (BALB/cOlaHsd-Foxn1 ). Subcutaneous xenograft tumors of taken every 3 hours using a 10 objective. IncuCyte software was HEK293 expressing FLAG-tagged human XIAP and caspase-9 were used to calculate mean percent confluency from four nonover- prepared as described previously (27). Tumors were measured lapping phase-contrast images of each well. using digital calipers, and volumes were calculated by applying Induction of apoptosis was measured over the first 24 hours by the formula for ellipsoid. including the Essen BioScience IncuCyte Caspase-3/7 Reagent at a For tumor growth inhibition studies, tumor-bearing animals final concentration of 2 mmol/L in all the wells. Apoptotic cells were randomized into groups of 7 to 8 with the average tumor were identified by the appearance of green-labeled nuclei, and volume of 100 mm3 (31 or 33 days after MDA-MB-231 cell green fluorescence was measured in real time in the green FL1 injection and 19 days after A375 injection). Mice were random- channel of the IncuCyte ZOOM live cell microscope. ized and oral ASTX660 treatment started on day 1. Control animals received water. During the treatment period, tumors were Apoptosis cytometry assays measured at least twice a week, and the effect on body weight was After incubation of the cells with ASTX660 for the designated recorded daily where possible. Statistical analyses were performed length of time, cells were harvested by trypsinization, spun using GraphPad Prism version 6. The effects of treatments were down, and 100 mL FACS buffer (PBS þ 1% FBS) was added. compared using one-way ANOVA and two-way ANOVA with Cells were then added to a 96-well plate and 100 mLof2 Dunnett multiple comparisons test against vehicle control. Dif- CellEvent reagent (Thermo Fisher Scientific; 4 mmol/L in FACS ferences were deemed statistically significant when P < 0.05. buffer) was added. The plate was incubated in the dark for 30 minutes before measuring fluorescent stained cells in a Guava Analysis of tumor sample pharmacodynamic markers easyCyte HT cytometer (Millipore). Cleaved caspase-3 staining Xenograft tumor lysates were prepared by grinding the frozen was recorded in the FL1 channel, with unstained and DMSO tissue to a fine powder with a mortar/pestle under liquid nitrogen, controlwellsbeingusedtosetthe gated stained and unstained and then adding ice-cold lysis buffer [1% Triton X-100, 150 cell populations. mmol/L NaCl, 20 mmol/L TrisHCl pH 7.5, plus protease inhibitors (Roche), 50 mmol/L NaF and 1 mmol/L Na3V04], to the Cell line viability screening ground-up tumor powder. Samples were vortexed and left on ice In-house cell viability assays were set up using alamarBlue for 30 minutes. Lysates were cleared by centrifugation, and reagent (Bio-Rad) as described previously (27). A human mela- samples of the supernatant removed for protein determination noma cell line panel was analyzed at ChemPartner by CellTiter- by BCA assay (Pierce). Glo luminescent cell proliferation assay (Promega) after ASTX660 For Western blotting, equivalent amounts of protein lysate had treatment for 72 hours in the presence or absence of 1 ng/mL SDS sample buffer and a final concentration of 50 mmol/L DTT TNFa (R&D Systems). Data were normalized to 0.1% DMSO (v/v) added, before being boiled, and analyzed by Western blotting as control, and the drug response, measured as the area over the described above. dose–response curve (activity area), was determined for each cell For immunoprecipitation assay of tumor lysates, equivalent line (29). amounts of xenograft lysate were incubated overnight at 4C with protein A/G magnetic beads (Pierce) coated with anti-XIAP In vivo studies polyclonal antibody (R&D Systems) followed by Western blot All mice were purchased from Envigo. The care and treatment analysis. For the MSD assay, equivalent amounts of xenograft of animals were in accordance with the United Kingdom Coor- lysate (200 mg/well) were incubated overnight at 4C in a strep- dinating Committee for Cancer Research guidelines and with tavidin MSD plate coated with biotinylated anti-XIAP polyclonal the United Kingdom Animals (Scientific Procedures) Act 1986 antibody (R&D Systems), before washing and probing with anti- (30, 31). The study protocols were approved by the University of SMAC, anti–caspase-9, or anti-XIAP antibody (Cell Signaling Cambridge Ethical Review Committee. Technology) followed by the appropriate secondary detection Initial pharmacokinetic studies were performed in male BALB/c antibody. wild type as described previously (27). ASTX660 was either dissolved in saline and administered intravenously at 5 mg/kg Pharmacokinetic analysis in a dose volume of 5 mL/kg or dissolved in water adjusted to pH Compound levels in plasma and tumor samples were mea- 5.5 with NaOH and administered by oral gavage at 5 to 30 mg/kg sured and pharmacokinetic parameters were calculated as in 10 mL/kg. Blood samples were collected at various time points described previously with the exception of sample bioanalysis, and plasma prepared by centrifugation. Further pharmacokinetic which was undertaken using reverse-phase liquid chromatogra- and pharmacodynamic studies were performed using tumor- phy-mass spectrometry (MS), using a Qtrap 4000 MS (AB Sciex), bearing immunocompromised animals (see below). Tumors coupled to an Agilent 1200 HPLC system (Agilent) or a Quattro were excised at specific time points after oral dose and immedi- Premier MS coupled to an Acquity UPLC system (Waters; ref. 27).

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Figure 1. ASTX660 is a novel antagonist of cIAP1/2 and XIAP. A, Chemical structure of non-peptidomimetic cIAP1/2 and XIAP antagonist, ASTX660, derived by fragment- based drug discovery. B and C, X-ray crystal structure of ASTX660 (in green) in complex with XIAP-BIR3 (PDB 5OQW). The Connolly surface of the protein in B is colored by electrostatic potential (red, negative; blue, positive; gray, neutral). Hydrogen bonds between ligand and protein in C are shown as dashed red lines.

Results XIAP and caspase-9 was potently inhibited with an EC50 value of 2.8 nmol/L (Fig. 2A; Supplementary Table S1). To confirm the ASTX660 is a novel IAP antagonist that targets the BIR3 domain antagonism of endogenous XIAP by ASTX660, we also investi- of cIAP1/2 and XIAP gated the displacement of SMAC from XIAP in A375, melanoma A fragment screen (28), subsequent structure-based drug design cells. Cell lysates, treated overnight with a range of ASTX660 campaign (27), and further optimization yielded ASTX660 (Fig. concentrations, were immunoprecipitated with anti-XIAP. A clear 1A), a potent, non-peptidomimetic, orally bioavailable, antago- reduction in SMAC levels immunoprecipitated with XIAP was nist of cIAP1/2 and XIAP. ASTX660 potently inhibited the inter- observed on treatment with concentrations of ASTX660 above actions between a SMAC-derived peptide and the BIR3 domains 0.01 mmol/L (Fig. 2B). Furthermore, exposure times as short as 5 of XIAP (BIR3-XIAP) and cIAP1 (BIR3-cIAP1) with IC values less 50 minutes to 1 mmol/L ASTX660 were sufficient to antagonize the than 40 and 12 nmol/L, respectively. The X-ray crystal structure of interaction of SMAC with XIAP in A375 cells (Fig. 2C). ASTX660 bound to BIR3-XIAP protein (PDB 5OQW) revealed that this inhibitor binds to the surface of the protein by occupying the same 4 pockets (P1-P4) also recognized by the N-terminal ASTX660 binds to and leads to the degradation of cIAP1/2 and sequence of the endogenous ligand SMAC (Fig. 1B) (32, 33). The stabilization of NIK in cells piperazine ring occupies the P1 pocket, with the protonated Binding of an IAP antagonist to the BIR3 domain of cIAP1/2 nitrogen forming hydrogen bonds with the side chain of Glu314 induces a conformational change, which activates the protein's and the backbone carbonyl of Asp309. The methyl substituent is ubiquitin ligase activity from the C-terminal RING domain (34). in van der Waals contact with the side chain of Trp310 and hence This leads to rapid autoubiquitination and proteasomal degra- efficiently fills a small lipophilic subpocket in P1. The P2 pocket is dation of cIAP1/2 (14). To demonstrate the antagonism of cIAPs occupied by the morpholine ring, which stacks on the top of the by ASTX660, levels of cIAP1 were measured in the human breast central amide. The carbonyl of the central amide forms a hydrogen cancer cell line, MDA-MB-231, after treatment with ASTX660. bond with the backbone NH of Thr308. ASTX660 extends into the Measurement of cIAP1 using an immunosorbent assay MSD P3 pocket with a 4-azaindoline bicycle, which forms further van detection platform demonstrated that ASTX660 induced degra- der Waals contacts with the side chain of Trp323, the backbone dation of cIAP1, with an EC50 of 0.22 nmol/L after 2 hours of carbonyl of Gly306, and the phenolic oxygen of the side chain of treatment (Fig. 3A; Supplementary Table S1). Further investiga- Tyr 324. Finally, the benzylic substituent grows from C-6 of the tion demonstrated a similar effect in A375 cells where cIAP1 levels azaindoline into P4 (Fig. 1C). dropped rapidly after treatment with 1 mmol/L ASTX660 and remained significantly decreased for up to 48 hours after addition ASTX660 potently antagonizes XIAP in cells of the compound (Fig. 3B). The human diffuse large B-cell Given the potent binding of ASTX660 to the isolated BIR3 lymphoma cell line, WSU-DLCL2, which has high basal cIAP2, domain of XIAP, we investigated the ability of ASTX660 to was used to demonstrate the effect of ASTX660 treatment on antagonize the effects of XIAP in cells. Both caspases and SMAC antagonism of both cIAP1 and cIAP2 over a range of times and bind to the BIR3 domain of XIAP in cells and so should be concentrations (Fig. 3C). ASTX660 treatment of WSU-DLCL2 displaced by a XIAP antagonist. We measured the displacement cells leads to significant cIAP1 and cIAP2 degradation after 1 of caspase-9 and SMAC in cells treated with ASTX660. To measure and 4 hours. Levels of cIAP2 were restored by 24 hours possibly XIAP:caspase-9 binding, we generated a stably transfected due to resistance previously observed with IAP antagonists after HEK293 cell line in which full-length XIAP (FLAG tagged) and prolonged treatment in a high NF-kB signaling background caspase-9 are overexpressed. This enabled us to observe the (35). TNFa-induced levels of cIAP2 are also antagonized in association of caspase-9 with XIAP. Two hours after addition of A375 and SK-MEL-28 melanoma cell lines after ASTX660 ASTX660 to this engineered cell line, the association between treatment (see Fig. 4B).

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Figure 2. ASTX660 treatment leads to potent antagonism of XIAP in cells. A, An engineered HEK293 cell line overexpressing XIAP and caspase-9 was treated for 2 hours with indicated concentrations of ASTX660. XIAP:caspase-9 binding was measured by immunoprecipitation of FLAG-tagged XIAP and quantitation of XIAP- associated caspase-9 using an MSD plate–based assay. Results show the mean of duplicate values. B, A375 cells were treated for 16 hours with the indicated concentrations of ASTX660. Endogenous XIAP antagonism was measured by Western blotting cell lysates for SMAC levels bound to XIAP following immunoprecipitation with anti-XIAP (top). Total XIAP and SMAC levels were determined by Western blots of cell lysates before immunoprecipitation (bottom). C, A375 cell lysates were treated with 1 mmol/L ASTX660 for the indicated times. XIAP antagonism was measured by Western blotting for SMAC levels after immunoprecipitation with anti-XIAP (top). Total XIAP and SMAC levels were determined by Western blots of cell lysates before immunoprecipitation (bottom).

Degradation of cIAPs leads to the stabilization of NIK and of this cell line was reduced with an EC50 of 1.8 nmol/L (Sup- activation of the noncanonical NF-kB pathway. The effects of plementary Table S1), whereas cleavage of PARP and caspase-3, ASTX660 treatment on NIK levels and NF-kB signaling were also detected by Western blot 24 hours after treatment with ASTX660, investigated. On treatment of MDA-MB-231 cells with ASTX660, indicated apoptosis was induced by ASTX660 treatment (Fig. 3D). the rapid degradation of cIAP1 was accompanied by a concomitant These effects were investigated further by measuring cell viability increase in the levels of NIK at 2 hours, which remained increased at and induction of cleaved capsase-3 in real time. Loss of viability 6 hours (Fig. 3D). The effect of NIK stabilization on noncanonical was observed soon after treatment with 0.1 mmol/L ASTX660 (Fig. NF-kB signaling was further demonstrated by the depletion of NF- 4A), and this was reversed on addition of a neutralizing anti-TNFa kB2 p100 and the increase in p52 after 24and 48 hours of treatment antibody, indicating that the effects of ASTX660 were TNFa with ASTX660. In addition, although canonical NF-kBsignaling dependent and that this cell line produces autocrine TNFa levels markers [phospho-p65 or NF-kB1 (p105/p50)] remained largely sufﬁcient for this ASTX660-induced response. The loss of viability unaltered, levels of phospho-IkBa were rapidly increased posttreat- was paralleled by an induction of apoptosis indicated by an ment and remained above basal levels up to 48 hours after increase in levels of cleaved caspase-3, an effect that, again, was treatment (Fig. 3D). Together, these data suggest that ASTX660 reversed by the addition of the neutralizing anti-TNFa antibody binds to cIAP1 and 2, leading to their degradation and downstream (Fig. 4A; Supplementary Fig. S1). effects on NF-kB signaling and apoptosis induction. Induction of apoptosis was further studied in two melanoma cell lines, A375 and SK-MEL-28. Although addition of 1 mmol/L The mechanism by which ASTX660 induces apoptosis in cell ASTX660 to these cell lines led to a degradation of both cIAP1 and lines is TNFa dependent cIAP2, apoptosis was only observed in the presence of TNFa as Effects of ASTX660 treatment on cell viability and apoptosis demonstrated by an increase in the levels of cleaved PARP, were investigated further in MDA-MB-231 cells. Overall viability caspase-3, and caspase-9, again indicating the dependence on

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Figure 3. Antagonism of cIAP1 by ASTX660 leads to its degradation in cancer cells. A, MDA-MB-231 cells were treated with the indicated concentrations of ASTX660 for 2 hours, lysed, and relative levels of cIAP1 measured using an MSD plate–based assay. Results show the mean of duplicate values. B, A375 cells were treated with 1 mmol/L ASTX660 for the indicated times and cIAP1 or XIAP levels analyzed by SDS-PAGE followed by immunoblotting. b-Actin was used as an internal control. C, WSU-DLCL2 cells were treated with the indicated concentrations of ASTX660 for the indicated times. Levels of cIAP1 and cIAP2 were analyzed by SDS-PAGE followed by immunoblotting. D, MDA-MB-231 cells were treated with the indicated concentrations of ASTX660 for 2, 6, 24, and 48 hours, lysed, and equal amounts of total protein were then analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies.

TNFa (Fig. 4B; Supplementary Fig. S2). Activity of caspase-3, as ASTX660 is orally bioavailable in mice and demonstrates a marker of apoptosis, was further investigated in these mel- prolonged antagonism of XIAP and cIAP1 in vivo anoma cell lines by measuring the cleavage of a caspase-3 The pharmacokinetic parameters of ASTX660 were evaluated substrate using flow cytometry. Treatment with ASTX660 or following intravenous and oral administration to mice. Following TNFa (1 ng/mL) alone showed no effect on caspase-3 activity, intravenous administration of ASTX660 at 5 mg/kg to male in either the A375 or SK-MEL-28 cells; however, when both BALB/c mice, a moderate mean clearance of 41 mL/minute/kg treatments were combined, the activity of caspase-3 was greatly was observed with a large volume of distribution of 2.7 L/kg. increased at time points up to 72 hours, indicating induction of ASTX660 was orally bioavailable (34%) following administration apoptosis (Fig. 4C). at 10 mg/kg to mice. Effects of ASTX660 treatment on cellular viability were also ASTX660 distributed into MDA-MB-231 (Fig. 5A) tumor xeno- investigated in a wider panel of 33 melanoma cell lines (Fig. 4D). grafts. ASTX660 was detectable in tumors up to 168 hours after a Addition of TNFa increased or potentiated the inhibitory effect of single oral dose of 20 mg/kg, and AUClast was 129 mmol/L/h/mL. In ASTX660 for the majority of cell lines, but not necessarily for all, contrast, plasma exposure achieved an AUClast 14.3 mmol/L/h/mL suggesting that at least in these cell lines, sufficient levels of and concentrations were below the limit of detection after 48 endogenous TNFa are produced. NHDF cell viability was not hours. A rapid reduction in cIAP1 levels coupled with antagonism significantly altered by ASTX660 treatment (Fig. 4D). of the XIAP:SMAC interaction was detected from 30 minutes

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Figure 4. TNFa triggers ASTX660-induced apoptosis in cell lines. A, Viability (top) and apoptosis (bottom) were measured in breast cancer MDA-MB-231 cells after treatment with 0.1 mmol/L ASTX660 using IncuCyte ZOOM live cell imaging. Anti-TNFa antibody at concentrations of 1 or 10 mg/mL was added to neutralize TNFa in the cell supernatant to monitor effects on the cell survival. B, Melanoma cell lines, A375 and SK-MEL-28, were treated with 1 mmol/L ASTX660 plus or minus 1 ng/mL TNFa for 24 hours. Cells were then lysed, and equal amounts of total protein were analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies. C, A375 or SK-MEL-28 cells were treated with 1 mmol/L ASTX660 for 25 hours in the presence or absence of 1 ng/mL TNFa. Cleaved caspase-3 activity was measured by cytometry. D, Effect of 72-hour ASTX660 treatment on the viability of 33 melanoma cell lines [plus NHDF cells (), included as a control]. Data generated by CellTiter-Glo in triplicate, in the presence or absence of 1 ng/mL TNFa, were normalized to 0.1% DMSO (v/v) control, and the drug response, measured as the area over the dose–response curve (activity area), was determined for each cell line (29). and for up to 3 days following a single dose in the tumors in the ASTX660 causes growth inhibition of MDA-MB-231 and A375 same experiment (Fig. 5B). Further experiments with Western xenograft tumors in vivo blotting analyses confirmed rapid cIAP1 degradation in MDA- The dose- and schedule-dependent antitumor effects of MB-231 xenograft tumors after treatment and a concomitant ASTX660 were investigated in the MDA-MB-231 breast cancer induction of apoptosis within one hour as demonstrated by an xenograft model (Fig. 6A and B; Supplementary Fig. S4A and increase in cleaved PARP and cleaved caspase-3 (Fig. 5C). A S4B). Daily oral administration of ASTX660 at 5, 10, and decrease in association of XIAP and SMAC was also observed in 20 mg/kg significantly inhibited tumor growth (P < 0.05 from xenograft tumor tissue, again after one hour of treatment (Fig. days 15, 18, and 11, respectively; Fig. 6A). An intermittent 5D), indicating antagonism of XIAP by ASTX660. The antagonistic schedule (cycles of 7 consecutive days of dosing followed by effects on XIAP were investigated further in a tumor model derived 7 days of dose-holiday) at 20 mg/kg also achieved significant from HEK293 cells genetically engineered to express XIAP and tumor growth inhibition, and its effects were equivalent to the caspase-9 (Supplementary Fig. S3). In this model, a single dose of continuous schedule in the same model (both P < 0.05 vs. ASTX660 disrupted the interaction between XIAP and caspase-9 vehicle treatment from day 8; Fig. 6B). The activity of ASTX660 or SMAC for at least 3 days. Tumor ASTX660 concentrations was further investigated in an A375 melanoma xenograft persisted over the duration of the study. model in nude mice (Fig. 6C; Supplementary Fig. S4C-i). Daily

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A C Vehicle control 1h 6 h 24 h 10,000 Plasma cIAP1

1,000 Tumor XIAP

100 Cleaved PARP

10 Cleaved Caspase-3

Mean ASTX660 (nmol/L) ASTX660 Mean concentraon 1 0 24487296120144168 SMAC Time aer single dose (h)

β-Acn

BD 110 110 Vehicle control 1 h 6 h 24 h 100 100 SMAC 90 XIAP:SMAC (IP) 90 80 cIAP1 Level 80 70 70 XIAP 60 60 50 50

40 40 Level cIAP1 (% vehicle control) (% vehicle control) 30 30 XIAP:SMAC Binding XIAP:SMAC 20 20 10 10 0 0 0 24487296120144168 Time (h)

Figure 5. Orally administered ASTX660 treatment modulates pharmacodynamic markers in a MDA-MB-231 xenograft model. A, SCID mice bearing MDA-MB-231 xenograft were treated with a single 20 mg/kg dose of ASTX660 and compound concentrations in plasma or tumor samples analyzed. B, In the same tumor samples, levels of cIAP1 and XIAP:SMAC association were determined by MSD assay. C, Mice bearing MDA-MB-231 xenograft tumors received a single oral dose of ASTX660 at 30 mg/kg. Animals were sacriﬁced at the indicated time points, and protein levels in tumors were measured by immunoblotting of whole-cell lysates. D, In the same tumor lysates, XIAP:SMAC association was analyzed by anti-XIAP immunoprecipitation. Each sample represents individual animals. MSD assay values represent mean SEM from 3 animals.

oral administration of ASTX660 at 10 and 20 mg/kg caused Discussion significant tumor growth inhibition in an A375 model (both P < 0.05 from day 8), although in vitro,thiscelllinewasonly Antagonism of IAPs is considered a promising therapeutic sensitivetoASTX660inthepresenceofTNFa in vitro (Fig. 4B approach for activating apoptosis pathways in cancer, and a and C). number of IAP antagonists have undergone evaluation in early In all three studies, the ASTX660 treatments were well tolerated, clinical trials (38). However, despite several observations of and no excessive bodyweight loss or significant adverse effects objective clinical response, single-agent activity in the clinic has were observed (Supplementary Fig. S4A-i, S4B-I, and S4C-i). It has been limited (39). The N-terminal sequence (AVPI) of the endog- fi been suggested that antagonizing multiple IAPs may lead to enous protein SMAC provided the starting point for the rst reduced tolerability in vivo (20, 36). To investigate the inflamma- generation of peptidomimetic IAP antagonists. The AVPI peptide tory effects of ASTX660, we tested the compound ex vivo on shows cIAP1 selectivity ( 200-fold over XIAP) due to differential peripheral blood mononuclear cells (PBMC) from 2 healthy interactions formed by the alanine residue in the P1 pocket of the human donors. We observed no substantial evidence of increase BIR3 domains of XIAP and cIAP1. We have shown that this fi in secretion of proinflammatory cytokines (TNFa, IL1b, IL2, IL6, intrinsic selectivity is retained in the rst generation of SMAC IL8, IFNg, and MCP-1) in a multiplexed cytokine assay (MSD) mimetics currently in the clinic (27). Given its function in inhibit- after a 48-hour treatment with 10 mmol/L of ASTX660 or with ing caspase activation, it has been proposed that XIAP antagonism 10 mmol/L LCL161 (see Supplementary Table S2). This was in plays a key role in the activation of apoptosis through both the contrast to the bivalent antagonist, BV-6 (Genentech; ref. 14), intrinsic and extrinsic pathways (2, 40). An IAP antagonist with fi which causes rapid cIAP1/2 depletion, potently antagonizes XIAP increased XIAP potency may lead to improved ef cacy due to (37), and induced cytokine elevation in the same PBMC assays more effective activation of apoptosis provided by releasing the (Supplementary Table S2). XIAP block on caspases (38, 45). An increased therapeutic

OF8 Mol Cancer Ther; 17(7) July 2018 Molecular Cancer Therapeutics

ASTX660 is an Antagonist of cIAP1/2 and XIAP

AB Vehicle control Vehicle control 700 600 20 mg/kg ASTX660 20 mg/kg ASTX660 q.d. )

600 10 mg/kg ASTX660 3 500 ) 3 5 mg/kg ASTX660 500 20 mg/kg ASTX660 q.d. 400 x 7/no dose x 7 400 300 300 200

200 (mm volume Tumor Tumor volume (mm volume Tumor 100 100

0 0 0 7 14 21 28 0 7 14 21 28 Day Day

C 800 Vehicle control 20 mg/kg ASTX660 ) 700 3 10 mg/kg ASTX660 600 500 400 300

Tumor volume (mm Tumor volume 200 100 0 0714 Day

Figure 6. Orally administered ASTX660 inhibits growth of MDA-MB-231 and A375 xenograft tumors. Each data point represents mean SEM from 8 animals unless otherwise indicated. A, ASTX660 was orally administered to SCID mice, bearing MDA-MB-231 xenografts, at 20, 10, and 5 mg/kg once daily for 25 days. Control animals received water. B, Comparison of the effects of varying ASTX660 administration schedule on MDA-MB-231 tumors. One group of tumor-bearing animals was treated with ASTX660 for 28 consecutive days (q.d., n ¼ 7), and another was given 2 cycles of 7 consecutive days of dosing followed by 7 days of dose- holiday (q.d. 7/no dose 7). On dosing days, both treatment groups received ASTX660 once daily at 20 mg/kg. The unconnected symbols represent the dosing events (red square: q.d. and blue triangle: q.d. 7/no dose 7). The control group received water as vehicle once daily for 25 consecutive days. C, A375-bearing nude mice were orally treated with 10 or 20 mg/kg of ASTX660 for 14 consecutive days.

window may also be achieved between efficacious dose and the approaches based on the AVPI tetrapeptide (27, 28). A subsequent onset of cytokine-induced toxicity that results from cIAP antag- lead optimization campaign focused on reducing off-target activ- onism. Cytokine release syndrome has been reported as the dose- ities and improving pharmacokinetic properties, resulting in limiting toxicity in a phase I dose escalation study of cIAP-selective ASTX660, a potent nonpeptidic IAP antagonist, structurally dis- antagonist LCL161 (23), and such effects have also been described tinct from IAP antagonists previously reported. The resulting IAP in detail in preclinical toxicity studies with another cIAP1/2 antagonism of ASTX660 was demonstrated both in vitro and antagonist GDC-0152 (41). A true antagonist of cIAP1/2 and in vivo. ASTX660 binds to both cIAP1 and XIAP with low nano- XIAP could better explore the clinical potential of IAP antagonism. molar potency, and this inhibition translates to cells, where To develop such a compound, we have applied our fragment- potent cIAP1/2 antagonism was demonstrated by their degrada- based drug discovery approach (Pyramid) to specifically tion, which occurs on the induction of autoubiquitinylation upon identify nonalanine leads with a cIAP1–cIAP2–XIAP profile, antagonist binding; at the same time, antagonism of XIAP was avoiding the intrinsic selectivity resulting from peptidomimetic measured by displacement of SMAC and casapse-9. Moreover, our

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Ward et al.

data also confirm here that ASTX660 has potent long-lasting ASTX660 is currently being tested in a phase I–II study in antagonism of cIAP1 and XIAP in vivo following oral administra- subjects with advanced solid tumors and lymphomas tion accompanied by significant inhibition of xenograft tumor (NCT02503423). Further studies with this novel compound will growth. In the assays used here, ASTX660 demonstrated potencies allow the true clinical potential of an IAP antagonist to be of 2.8 and 0.22 nmol/L, respectively, for XIAP and cIAP1 antag- explored both as a single agent and in combination. onism (12.7-fold difference). In contrast, other clinical stage monovalent SMAC mimetics do not achieve this level of potency Disclosure of Potential Conflicts of Interest for XIAP (range, 10–35 nmol/L) or this balance of antagonism G.A. Ward, E.J. Lewis, J.S. Ahn, C.N. Johnson, J.F. Lyons, V. Martins, (39-227 fold difference) under the same conditions (see Supple- J.M. Munck, S.J. Rich, T. Smyth, N.T. Thompson, P.A. Williams, N.E. Wilsher, mentary Table S1), making ASTX660 a novel compound for N.G. Wallis, and G. Chessari are employees of Astex Pharmaceuticals. further investigating the effects of IAP antagonism. There are several potential advantages to antagonizing XIAP in addition to cIAP1/2. Aberrantly high levels of XIAP have been Authors' Contributions linked to poor prognosis in a number of tumor types, including Conception and design: G.A. Ward, J.F. Lyons, N.T. Thompson, N.G. Wallis, diffuse large B-cell lymphoma, renal cell carcinoma, bladder G. Chessari – Development of methodology: G.A. Ward, E.J. Lewis, C.N. Johnson cancer, and colorectal cancer (42 45), while overexpression of Acquisition of data (provided animals, acquired and managed patients, XIAP in response to chemotherapy and radiotherapy has been provided facilities, etc.): G.A. Ward, E.J. Lewis, J.S. Ahn, C.N. Johnson, proposed to contribute to resistance to these treatments (42, 46, V. Martins, J.M. Munck, S.J. Rich, P.A. Williams, N.E. Wilsher 47). In addition, XIAP levels have also been implicated in mod- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, ulating responses to immunotherapy, with increased levels in computational analysis): G.A. Ward, E.J. Lewis, J.S. Ahn, C.N. Johnson, Hodgkin lymphoma preventing cytotoxic lymphocyte–mediated V. Martins, J.M. Munck, S.J. Rich, T. Smyth, N.T. Thompson, P.A. Williams, – N.E. Wilsher, N.G. Wallis, G. Chessari cytotoxicity by blocking granzyme B induced apoptosis (48) and Writing, review, and/or revision of the manuscript: G.A. Ward, C.N. Johnson, upregulation of XIAP in inflammatory breast cancer driving J.F. Lyons, V. Martins, T. Smyth, P.A. Williams, N.E. Wilsher, N.G. Wallis, resistance to antibody-dependent cell-mediated cytotoxicity G. Chessari (49), further indicating potential for a molecule that potently Administrative, technical, or material support (i.e., reporting or organizing antagonizes XIAP. Furthermore, the role of XIAP in both extrinsic data, constructing databases): G.A. Ward, V. Martins and intrinsic apoptosis suggests that a potent XIAP inhibitor, such Study supervision: J.F. Lyons, N.T. Thompson as ASTX660, might also be expected to show increased synergy with activators of both these apoptotic pathways, such as che- Acknowledgments motherapeutics, radiotherapy, or TRAIL agonists, compared with We warmly acknowledge scientific discussions with many Astex colleagues. a more cIAP-selective antagonist (2). It has been suggested that We also thank Anne Cleasby for critical discussions and crystallographic certain bivalent compounds that potently antagonize cIAP and support. XIAP are not well tolerated in vivo as they induce an excessive The costs of publication of this article were defrayed in part by the payment of cytokine-mediated proinflammatory phenotype (20, 36). How- page charges. This article must therefore be hereby marked advertisement in ever, we have demonstrated that, unlike the bivalent compounds, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ASTX660 does not elicit a toxic cytokine signature, suggesting that the IAP antagonist properties of this compound are not a disad- Received September 8, 2017; revised January 4, 2018; accepted April 16, 2018; vantage in this respect. published first April 25, 2018.

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www.aacrjournals.org Mol Cancer Ther; 17(7) July 2018 OF11

ASTX660, a Novel Non-peptidomimetic Antagonist of cIAP1/2 and XIAP, Potently Induces TNF α-Dependent Apoptosis in Cancer Cell Lines and Inhibits Tumor Growth

George A. Ward, Edward J. Lewis, Jong Sook Ahn, et al.

Mol Cancer Ther Published OnlineFirst April 25, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-17-0848

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