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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

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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. Ó2018 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 Ó2018 American Association for Cancer Research. the presence of TNFa via the extrinsic apoptosis pathway. Tumors www.aacrjournals.org OF1 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst April 25, 2018; DOI: 10.1158/1535-7163.MCT-17-0848 Ward et al. 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-
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