ASTX660, a novel non-peptidomimetic antagonist of cIAP1/2 ... · by fragment-based screening and...

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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

Authors: 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, Gianni Chessari.

Affiliation: Astex Pharmaceuticals, 436 Cambridge Science Park, Milton Road, Cambridge, CB4 0QA, UK.

Running title: ASTX660 is an antagonist of cIAP1/2 and XIAP

Key Words: XIAP, cIAP1, cIAP2, apoptosis, ASTX660, antagonist

Authors to whom correspondence should be addressed:

George A Ward, Astex Pharmaceuticals, 436 Cambridge Science Park, Milton Road, Cambridge, CB4 0QA,

UK. Tel: +44 1223 435068, Fax: +44 1223 226201, Email: [email protected]

Conflict of Interest Statement: GAW, EJL, JSA, CNJ, JFL, VM, JMM, SJR, TS, NTT, PAW, NEW, NGW, GC

are employees of Astex Pharmaceuticals

Total word count: 4902, Total Figures and Tables: 6

on March 29, 2020. © 2018 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 25, 2018; DOI: 10.1158/1535-7163.MCT-17-0848

mailto:[email protected]

http://mct.aacrjournals.org/

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Abstract

Due to their roles in the evasion of apoptosis, Inhibitor of Apoptosis Proteins (IAPs) are considered

attractive targets for anti-cancer therapy. Antagonists of these proteins have the potential to switch pro-

survival signaling pathways in cancer cells towards cell death. Various SMAC-peptidomimetics with

inherent cIAP selectivity have been tested clinically and demonstrated minimal single agent efficacy.

ASTX660 is a potent, non-peptidomimetic, antagonist of cIAP1/2 and XIAP, discovered using fragment-

based drug design. The antagonism of XIAP and cIAP1 by ASTX660 was demonstrated on purified

proteins, cells and in vivo in xenograft models. The compound binds to the isolated BIR3 domains of

both XIAP and cIAP1 with nanomolar potencies. In cells and xenograft tissue direct antagonism of XIAP

was demonstrated by measuring its displacement from caspase-9 or SMAC. Compound-induced

proteasomal degradation of cIAP1 and 2, resulting in downstream effects of NIK stabilization and

activation of non-canonical NF-B signaling, demonstrated cIAP1/2 antagonism. Treatment with

ASTX660 led to TNF--dependent induction of apoptosis in various cancer cell lines in vitro, while dosing

in mice bearing breast and melanoma tumor xenografts inhibited tumor growth. ASTX660 is currently

being tested in a phase 1-2 clinical trial (NCT02503423) and we propose that its antagonism of cIAP1/2

and XIAP may offer improved efficacy over first-generation antagonists that are more cIAP1/2 selective.




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Introduction

Evasion of apoptosis is one of the hallmarks of cancer (1) and can be achieved by overexpression of anti-

apoptotic proteins. Inhibitor of apoptosis proteins (IAPs), such as cellular IAP (cIAP) 1 and 2 and X-linked

IAP (XIAP), are key regulators of anti-apoptotic and pro-survival signaling pathways; XIAP directly inhibits

caspases, whilst cIAPs prevent the formation of pro-apoptotic signaling complexes. This leads to

suppression of apoptosis through both the extrinsic and intrinsic apoptosis pathways (2-4). Their

deregulation, through amplification, overexpression or loss of endogenous antagonists, occurs in various

cancers and is associated with tumor growth and poor prognosis, making them attractive targets for

anti-cancer therapy (5).

The IAPs are characterized by their baculovirus IAP repeat (BIR) domains which mediate protein:protein

interactions; some members of the family such as cIAP and XIAP also possess RING (Really Interesting

New Gene) zinc finger domains with E3 ubiquitin ligase activity (6,7). The anti-apoptotic activity of XIAP

is mediated by its direct binding to and inactivation of caspases 3, 7 and 9 via its BIR domains (8). IAP

antagonists such as the endogenous second mitochondria derived activator of caspases (SMAC), which is

released from mitochondria on induction of apoptosis, bind to the BIR domains of IAPs and can disrupt

interactions such as those between XIAP and caspase 9 (9). On binding to other IAPs (cIAP1 and cIAP2),

SMAC induces a conformational change, which activates their E3 ligase function leading to rapid

autoubiquitination and proteasomal degradation (10).

In response to TNF-α, cIAPs ubiquitinate RIP1 promoting the formation of complexes (e.g. complex I),

which activate survival signaling through the canonical NF-B pathway. Simultaneously, formation of

the death-inducing signaling complex (DISC), which drives apoptosis, is prevented. Antagonism and

subsequent degradation of the cIAP1/2 leads to the stabilization of NIK (NF-B-inducing kinase) which

activates the non-canonical NF-B pathway, resulting in the production of multiple cytokines including

TNF-α. Removal of the cIAP1/2 also allows the DISC to form, leading overall to a switch in TNF-α

signaling from pro-survival to pro-apoptotic (11-15). This loss of cIAP1/2 combined with release of the

XIAP-mediated block on caspases, which is essential for full activation of apoptosis, leads to a sustained

pro-apoptotic effect in the presence of TNF-α via the extrinsic apoptosis pathway. Tumors with

sufficient levels of TNF-α in their environment may, therefore, be particularly susceptible to IAP

antagonism (16). In addition, the antagonism of XIAP-mediated caspase inhibition promotes apoptosis

induced by stimulation of the intrinsic apoptosis pathway by agents such as chemotherapeutics or DNA




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damaging agents (17). This suggests that cIAP1/cIAP2/XIAP antagonists can be used to promote

apoptosis through both the extrinsic and intrinsic pathways.

IAPs have been therapeutically targeted by antisense oligonucleotides and antagonist small molecules

(4). AEG35156, an antisense oligonucleotide targeted to XIAP, showed some evidence of clinical activity

(18) and sensitized cancer cells to chemotherapeutic agents and TRAIL receptor agonists in preclinical

models (3). The first generation of SMAC-mimetic IAP antagonists to enter the clinic, all containing

alanine moieties and inherently cIAP-selective, have shown some activity in preclinical models (19-22) ,

but thus far limited single agent efficacy in clinical trials (3,4,23-26).

We have previously reported the identification of lead compounds with activity against cIAP1 and XIAP

by fragment-based screening and structure-based drug design (27,28). Here we describe the discovery

and characterization of ASTX660, an antagonist of cIAP1/2 and XIAP, which is currently being tested in a

phase 1-2 clinical trial (NCT02503423). We hypothesize that such IAP antagonism may lead to improved

efficacy as a result of the more effective activation of apoptosis provided by blocking cIAP1/2 while

releasing the XIAP-block on caspases.




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Materials and Methods

Materials

ASTX660 as the hydrochloride salt was synthesized using a chemical procedure similar to that used for

AT-IAP (27). The key step involved the coupling reaction between methyl-5-((R)-3-methyl-morpholin-4-

ylmethyl)-piperazine-1-carboxylic acid tert-butyl ester and 2-chloro-1-{6-[(4-fluorophenyl)methyl]-5-

(hydroxymethyl)-3,3-dimethyl-1H,2H,3H-pyrrolo[3,2-b]pyridin-1-yl}ethan-1-one (see Supplementary

Materials and Methods); purity was determined as greater than 95% by high-performance liquid

chromatography. All other reagents were purchased from Sigma (Poole, UK) unless otherwise stated.

BV-6 [(2S)-2-{[(2S)-1-[(2S)-2-cyclohexyl-2-[(2S)-2-(methylamino)propanamido]acetyl]pyrrolidin-2-

yl]formamido}-N-{6-[(2S)-2-{[(2S)-1-[(2S)-2-cyclohexyl-2-[(2S)-2-

(methylamino)propanamido]acetyl]pyrrolidin-2-yl]formamido}-3,3-diphenylpropanamido]hexyl}-3,3-

diphenylpropanamide] (14) was purchased from Selleckchem (Munich, Germany).

Protein Production and crystallography

A XIAP-BIR3 construct (amino acids 250-354) was expressed in E. coli, purified by affinity and size-

exclusion column chromatography and crystallized at approximately 10 mg/mL as described previously

(27). Crystals were soaked with 5 mM ASTX660 in 5% DMSO overnight at room temperature prior to

data collection. The crystals had cell dimensions of approximately 70 Å × 70 Å × 105 Å and belong to

space group P4122. The diffraction observed ranged from 1.7 to 3.0 Å.

Binding assays

Interaction between ASTX660 and the BIR3 domains of XIAP or cIAP1 was determined by measuring the

displacement of a fluorescent peptide tracer derived from SMAC (AbuRPFK(5&6FAM)-amide; Peptide

Synthetics Ltd, Fareham, Hampshire, UK ) by fluorescence polarization on a Pherastar plate reader (BMG

Labtech, Orttenburg, Germany). IC50 curves were generated using GraphPad Prism version 6 (La Jolla,

CA, USA) and fitted using the four parameter logistic curve fit.




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Cell Lines

The human cell lines MDA-MB-231 and HEK293 were purchased from the European Collection of Cell

Cultures (ECACC) (Porton Down, UK); human melanoma cell lines, A375 and SK-MEL-28, were purchased

from American Type Culture Collection (ATCC) (Teddington, UK); and the diffuse large B-cell lymphoma

cell line, WSU-DLCL2, was purchased from DSMZ (Braunschweig, Germany). All were grown in DMEM

medium supplemented with 10% FBS and maintained at 37 oC in an atmosphere of 5% CO2 except WSU-

DLCL2 cells which were grown as above except in RPMI medium supplemented with 10% FBS. All cell

culture reagents were purchased from Invitrogen unless stated otherwise. These cells lines were not

passaged for more than 6 months (or 30 passages) after authentication by the cell bank (short tandem

repeat PCR), and were routinely screened for mycoplasma (MycoAlert, LONZA, Basel Switzerland).

Melanoma cell lines screened at ChemPartner (Shanghai, China) were purchased from ATCC, except

COLO679 (ECACC), GAK (Japanese Collection of Research Bioresources, Osaka, Japan), MMAC-SF (Riken

Cell Bank, Tsukuba, Japan) and NHDF (LONZA); and were mycoplasma screened, short tandem repeat

PCR verified, and not used beyond 10 passages.

Western blots

Cell lysate samples were resolved by SDS-PAGE and immunoblotted as described previously (27) with

antibodies specific for XIAP (AF8221), cIAP1 (AF8181) and cIAP2 (AF8171) from R&D Systems (Abingdon,

UK); and the following antibodies from Cell Signaling Technology (Hitchin, UK): cleaved caspase-3

(#9664), cleaved PARP (#9541), cleaved caspase-9 (#9505), phospho-p65 (S536) (#3033), phospho-IB

(Ser32) (#2859), total IB (#4814), NF-B1 (p105/p50) (#3035), NF-B2 (p100/p52) (#4882), SMAC

(#2954), NIK (4994) and -actin (#8457).

Immunoprecipitation with Anti-XIAP

Equivalent amounts of cell lysate were incubated overnight at 4 °C with protein A/G magnetic beads

(Pierce, Paisley, UK) coated with anti-XIAP polyclonal antibody (R&D Systems). The beads were washed

and boiled in SDS sample buffer containing DTT, before analysis of the eluted proteins by Western

blotting. Western blots of the same lysate before immunoprecipitation were used for comparison.

Antagonism of XIAP by ASTX660 was monitored by Western blotting for levels of SMAC

immunoprecipitated by XIAP.




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Mesoscale Discovery Assays

An engineered HEK293 cell line (clonal isolate from stable transfection with full-length FLAG-tagged XIAP

expression construct (RC207627) and a full-length untagged caspase-9 construct (SC119362) (Origene,

Rockville, USA)) were used to detect XIAP binding to caspase-9. Cells were incubated with compound for

2 hours, lysed and lysates added to streptavidin Mesoscale Discovery (MSD) plates coated with

biotinylated anti-XIAP polyclonal antibody (R&D Systems), before washing and probing with anti-

caspase-9 or anti-XIAP antibody (Cell Signaling Technology) followed by the appropriate secondary

detection antibody.

A Mesoscale Discovery (MSD) plate-based assay was used to quantify levels of cIAP1 in MDA-MB-231

after two hour ASTX660 treatment, cells. Cells were incubated with compound for two hours, washed

and lysed. Lysates were applied to MSD plates as described previously (27).

Live cell imaging

Cells were imaged in real-time using the IncuCyte ZOOM live cell imager (Essen BioScience, Ann Arbor,

Michigan). Cells were incubated with compound in 0.1% (v/v) dimethyl sulfoxide (DMSO), with or

without neutralizing anti-TNF- antibody (R&D Systems) for 5 days and live images were taken every 3

hours using a 10× objective. IncuCyte software was used to calculate mean percent confluency from

four non-overlapping phase contrast images of each well.

Induction of apoptosis was measured over the first 24 hours by including the Essen BioScience

IncuCyte™ Caspase-3/7 Reagent at a final concentration of 2 µM in all the wells. Apoptotic cells were

identified by the appearance of green-labelled nuclei, and green fluorescence was measured in real-time

in the green FL1 channel of the IncuCyte ZOOM live cell microscope.

Apoptosis Cytometry Assays

After incubation of the cells with ASTX660 for the designated length of time, cells were harvested by

trypsinisation, spun down and 100 µl FACS buffer (PBS + 1% FBS) added. Cells were then added to a 96-

well plate and 100 µl of 2 X CellEvent reagent (Thermo, Paisley, UK) (4 µM in FACS buffer) was added.

The plate was incubated in the dark for 30 minutes before measuring fluorescent stained cells in a

Guava easyCyte HT cytometer (Millipore, Billerica, Massachusetts). Cleaved caspase-3 staining was

recorded in the FL1 channel, with unstained and DMSO control wells being used to set the gated stained

and unstained cell populations.




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Cell Line Viability Screening

In-house cell viability assays were set up using alamarBlue reagent (Bio-Rad, Oxford, UK) as described

previously (27). A human melanoma cell line panel was analysed at ChemPartner (Shanghai, China) by

CellTiter-Glo luminescent cell proliferation assay (Promega, Madison, Wisconsin) after ASTX660

treatment for 72 hours in the presence or absence of 1 ng/mL TNF- (R&D Systems). Data were

normalised 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).

In vivo Studies

All mice were purchased from Envigo (Bicester, UK). The care and treatment of animals were in

accordance with the United Kingdom Coordinating Committee for Cancer Research guidelines and with

the United Kingdom Animals (Scientific Procedures) Act 1986 (30,31). The study protocols were

approved by the University of Cambridge Ethical Review Committee.

Initial pharmacokinetic studies were performed in male BALB/c wild type as described previously (27).

ASTX660 was either dissolved in saline and administered intravenously (IV) at 5 mg/kg in a dose volume

of 5 mL/kg or dissolved in water adjusted to pH5.5 with NaOH and administered by oral gavage (PO) at 5

to 30 mg/kg in 10 mL/kg. Blood samples were collected at various time points and plasma prepared by

centrifugation. Further pharmacokinetic and pharmacodynamic studies were performed using tumor-

bearing immunocompromised animals (see below). Tumors were excised at specific time points post

oral dose and immediately snap-frozen in liquid nitrogen before being stored at – 80 °C prior to analysis.

MDA-MB-231 xenografts were prepared by subcutaneously injecting 5x106 cells, suspended in 100 µl of

serum-free medium, into the right hind flank of male severe combined immunodeficient (SCID,

BALB/cJHan®Hsd-Prkdcscid) mice. A375 xenografts were prepared by subcutaneously injecting 5x106

cells, suspended in 100 µl of a 1:1 mixture of serum-free medium and Matrigel (approximately 10

mg/mL, Corning), into the right hind flank of male nude mice (BALB/cOlaHsd-Foxn1nu). Subcutaneous

xenograft tumors of HEK293 expressing FLAG-tagged human XIAP and caspase 9 were prepared as

previously described (27). Tumors were measured using digital calipers and volumes calculated by

applying the formula for ellipsoid.

For tumor growth inhibition studies, tumor-bearing animals were randomized into groups of 7 to 8 with

the average tumor volume of 100 mm3 (31 or 33 days after MDA-MB-231 cell injection and 19 days after



http://www.envigo.com/products-services/research-models-services/models/research-models/mice/mutant/scid-mice/balb-cjhanahsd-prkdcscid/

http://www.envigo.com/products-services/research-models-services/models/research-models/mice/mutant/balb-c-nude-mice/balb-colahsd-foxn1nu/


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A375 injection). Mice were randomized and oral ASTX660 treatment started on Day 1. Control animals

received water. During the treatment period, tumors were measured at least twice a week and the

effect on body weight recorded daily where possible. Statistical analyses were performed using

GraphPad Prism version 6. The effects of treatments were compared using one-way ANOVA and two-

way ANOVA with Dunnett’s multiple comparisons test against vehicle control. Differences were deemed

statistically significant when P<0.05.

Analysis of Tumor Sample Pharmacodynamic Markers

Xenograft tumor lysates were prepared by grinding the frozen tissue to a fine powder with a

mortar/pestle under liquid nitrogen, and then adding ice-cold lysis buffer (1% Triton X-100, 150 mM

NaCl, 20 mM Tris·HCl pH 7.5, plus protease inhibitors (Roche, Burgess Hill, UK), 50 mM NaF and 1 mM

Na3V04), to the ground-up tumor powder. Samples were vortexed and left on ice for 30 min. Lysates

were cleared by centrifugation and samples of the supernatant removed for protein determination by

BCA assay (Pierce).

For Western blotting, equivalent amounts of protein lysate had SDS sample buffer and a final

concentration of 50 mM DTT added, before being boiled and analyzed by Western blotting as described

above.

For immunoprecipitation assay of tumor lysates, equivalent amounts of xenograft lysate were incubated

overnight at 4 °C with protein A/G magnetic beads (Pierce) coated with anti-XIAP polyclonal antibody

(R&D Systems) followed by western blot analysis. For the MSD assay, equivalent amounts of xenograft

lysate (200 µg/well) were incubated overnight at 4 °C in a streptavidin MSD plate coated with

biotinylated anti-XIAP polyclonal antibody (R&D Systems), before washing and probing with anti-SMAC,

anti-caspase-9 or anti-XIAP antibody (Cell Signaling Technology, Hitchin, UK) followed by the appropriate

secondary detection antibody.

Pharmacokinetic Analysis

Compound levels in plasma and tumor samples were measured and pharmacokinetic parameters

calculated as described previously with the exception of sample bioanalysis which was undertaken using

reverse-phase liquid chromatography-mass spectrometry (MS), using a Qtrap 4000® MS (AB Sciex, UK),

coupled to an Agilent 1200 HPLC system (Agilent, UK) or a Quattro Premier MS coupled to an Acquity

UPLC system (Waters, UK) (27).




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Results

ASTX660 is a novel IAP antagonist that targets the BIR3 domain of cIAP1/2 and XIAP

A fragment screen (28), subsequent structure-based drug design campaign (27) and further optimization

yielded ASTX660 (Figure 1A), a potent, non-peptidomimetic, orally-bioavailable, antagonist of cIAP1/2

and XIAP. ASTX660 potently inhibited the interactions between a SMAC-derived peptide and the BIR3

domains of XIAP (BIR3-XIAP) and cIAP1 (BIR3-cIAP1) with IC50 values less than 40 nM and 12 nM

respectively. The X-ray crystal structure of 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

recognised by the N-terminal sequence of the endogenous ligand SMAC (Figure 1B) (32,33). The

piperazine ring occupies the P1 pocket, with the protonated nitrogen forming hydrogen bonds with the

side chain of Glu314 and the backbone carbonyl of Asp309. The methyl substituent is in van der Waals

contact with the side chain of Trp310, and hence efficiently fills a small lipophilic sub-pocket in P1. The

P2 pocket is occupied by the morpholine ring which stacks on the top of the central amide. The carbonyl

of the central amide forms a hydrogen bond with the backbone NH of Thr308. ASTX660 extends into the

P3 pocket with a 4-azaindoline bicycle which forms further van der Waals contacts with the side chain of

Trp323, the backbone carbonyl of Gly306 and the phenolic oxygen of the side chain of Tyr 324. Finally,

the benzylic substituent grows from C-6 of the azaindoline into P4 (Figure 1C).

ASTX660 potently antagonises XIAP in cells

Given the potent binding of ASTX660 to the isolated BIR3 domain of XIAP, we investigated the ability of

ASTX660 to antagonize the effects of XIAP in cells. Both caspases and SMAC bind to the BIR3 domain of

XIAP in cells and so should be displaced by a XIAP antagonist. We measured the displacement of

caspase-9 and SMAC in cells treated with ASTX660. To measure XIAP:caspase-9 binding we generated a

stably transfected HEK293 cell line in which full length XIAP (FLAG tagged) and caspase-9 are over-

expressed. This enabled us to observe the association of caspase-9 with XIAP. Two hours after addition

of ASTX660 to this engineered cell line, the association between XIAP and caspase 9 was potently

inhibited with an EC50 value of 2.8 nM (Figure 2A, Supplementary Table 1). To confirm the antagonism of

endogenous XIAP by ASTX660 we also investigated the displacement of SMAC from XIAP in A375,

melanoma cells. Cell lysates, treated overnight with a range of ASTX660 concentrations, were immuno-

precipitated with anti-XIAP. A clear reduction in SMAC levels immunoprecipitated with XIAP was

observed on treatment with concentrations of ASTX660 above 0.01 µM (Figure 2B). Furthermore,




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exposure times as short as 5 minutes to 1 µM ASTX660 were sufficient to antagonise the interaction of

SMAC with XIAP in A375 cells (Figure 2C).

ASTX660 binds to and leads to the degradation of cIAP1/2 and stabilisation of NIK in cells

Binding of an IAP antagonist to the BIR3 domain of cIAP1/2 induces a conformational change which

activates the protein’s ubiquitin ligase activity from the C-terminal RING domain (34). This leads to rapid

auto-ubiquitination and proteasomal degradation of cIAP1/2 (14). In order to demonstrate the

antagonism of cIAPs by ASTX660, levels of cIAP1 were measured in the human breast cancer cell line,

MDA-MB-231, after treatment with ASTX660. Measurement of cIAP1 using an immunosorbent assay

MSD detection platform demonstrated that ASTX660 induced degradation of cIAP1, with an EC50 of 0.22

nM after two hours of treatment (Figure 3A, Supplementary Table 1). Further investigation

demonstrated a similar effect in A375 cells where cIAP1 levels dropped rapidly after treatment with 1

µM ASTX660 and remained significantly decreased for up to 48 hours after addition of compound

(Figure 3B). The human diffuse large B-cell lymphoma cell line, WSU-DLCL2, which has high basal cIAP2,

was used to demonstrate the effect of ASTX660 treatment on antagonism of both cIAP1 and cIAP2 over

a range of times and concentrations (Figure 3C). ASTX660 treatment of WSU-DLCL2 cells leads to

significant cIAP1 and cIAP2 degradation after 1 and 4 hours. Levels of cIAP2 were restored by 24 hours

possibly due to resistance previously observed with IAP antagonists after prolonged treatment in a high

NF-B signaling background(35). TNF--induced levels of cIAP2 are also antagonized in A375 and SK-

MEL-28 melanoma cell lines after ASTX660 treatment (see Figure 4B).

Degradation of cIAPs leads to the stabilization of NIK and activation of the non-canonical NF-B

pathway. The effects of ASTX660 treatment on NIK levels and NF-B signaling were also investigated.

On treatment of MDA-MB-231 cells with ASTX660, the rapid degradation of cIAP1 was accompanied by a

concomitant increase in the levels of NIK at 2 h which remained increased at 6 h (Figure 3D). The effect

of NIK stabilization on non-canonical NF-B signaling was further demonstrated by the depletion of NF-

B2 p100 and the increase in p52 after 24 and 48 h treatment with ASTX660. In addition, whilst

canonical NF-B signaling markers (phospho-p65 or NF-B1 (p105/p50)) remained largely unaltered,

levels of phospho-IB were rapidly increased post treatment and remained above basal levels up to 48

h after treatment (Figure 3D). Together these data suggest that ASTX660 binds to cIAP1 and 2 leading to

their degradation and downstream effects on NF-B signaling and apoptosis induction.

The mechanism by which ASTX660 induces apoptosis in cell lines is TNF--dependent




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Effects of ASTX660 treatment on cell viabilityand apoptosis were investigated further in MDA-MB-231

cells. Overall viability of this cell line was reduced with an EC50 of 1.8 nM (Supplementary Table 1),

whilst cleavage of PARP and caspase-3, detected by Western blot 24 h after treatment with ASTX660,

indicated apoptosis was induced by ASTX660 treatment (Figure 3D). These effects were investigated

further by measuring cell viabilityand induction of cleaved capsase-3 in real time. Loss of viability was

observed soon after treatment with 0.1 µM ASTX660 (Figure 4A) and this was reversed on addition of a

neutralising anti-TNF-antibody, indicating that the effects of ASTX660 were TNF-α dependent and that

this cell line produces autocrine TNF- levels sufficient for this ASTX660-induced response. The loss of

viability was paralleled by an induction of apoptosis indicated by an increase in levels of cleaved

caspase-3, an effect which, again, was reversed by the addition of the neutralising anti-TNF-antibody

(Figure 4A and Supplementary Figure 1).

Induction of apoptosis was further studied in two melanoma cell lines, A375 and SK-MEL-28. Whilst

addition of 1 µM ASTX660 to these cell lines led to a degradation of both cIAP1 and cIAP2, apoptosis was

only observed in the presence of TNF- as demonstrated by an increase in the levels of cleaved PARP,

caspase-3 and caspase-9, again indicating the dependence on TNF-α (Figure 4B, Supplementary Figure

2). Activity of caspase-3, as a marker of apoptosis, was further investigated in these melanoma cell lines

by measuring the cleavage of a caspase-3 substrate using flow cytometry. Treatment with ASTX660 or

TNF- (1 ng/mL) alone showed no effect on caspase 3 activity, in either the A375 or SK-MEL-28 cells,

however when both treatments were combined the activity of caspase-3 was greatly increased at time

points up to 72 h indicating induction of apoptosis (Figure 4C).

Effects of ASTX660 treatment on cellular viability was also investigated in a wider panel of 33 melanoma

cell lines (Figure 4D). Addition of TNF-α increased or potentiated the inhibitory effect of ASTX660 for

the majority of cell lines, but not necessarily for all, suggesting that at least in these cell lines sufficient

levels of endogenous TNF-α are produced. Normal human dermal fibroblast (NHDF) cell viability was

not significantly altered by ASTX660 treatment (Figure 4D).

ASTX660 is orally bioavailable in mice and demonstrates prolonged antagonism of XIAP and cIAP1 in

vivo

The pharmacokinetic parameters of ASTX660 were evaluated following IV and PO administration to

mice. Following IV administration of ASTX660 at 5 mg/kg to male BALB/c mice, a moderate mean




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clearance of 41 mL/min/kg was observed with a large volume of distribution of 2.7 L/kg. ASTX660 was

orally bioavailable (34%) following administration at 10 mg/kg to mice.

ASTX660 distributed into MDA-MB-231 (Figure 5A) tumor xenografts. ASTX660 was detectable in

tumors up to 168 h after a single oral dose of 20 mg/kg, and area under the curve (AUC)last was 129

µM∙h/mL. In contrast, plasma exposure achieved an AUClast 14.3 µM∙h/mL and concentrations were

below the limit of detection after 48h. A rapid reduction in cIAP1 levels coupled with antagonism of the

XIAP:SMAC interaction was detected from 30 min and for up to 3 days following a single dose in the

tumors in the same experiment (Figure 5B). Further experiments with Western blotting analyses

confirmed rapid cIAP1 degradation in MDA-MB-231 xenograft tumors after treatment and a

concomitant induction of apoptosis within one hour as demonstrated by an increase in cleaved PARP

and cleaved caspase-3 (Figure 5C). A decrease in association of XIAP and SMAC was also observed in

xenograft tumor tissue, again after one hour of treatment (Figure 5D), indicating antagonism of XIAP by

ASTX660. The antagonistic effects on XIAP were investigated further in a tumor model derived from

HEK293 cells genetically engineered to express XIAP and caspase-9 (Supplementary Figure 3). In this

model, a single dose of ASTX660 disrupted the interaction between XIAP and caspase-9 or SMAC for at

least 3 days. Tumor ASTX660 concentrations persisted over the duration of the study.

ASTX660 causes growth inhibition of MDA-MB-231 and A375 xenograft tumors in vivo.

The dose- and schedule-dependent anti-tumor effects of ASTX660 were investigated in the MDA-MB-

231 breast cancer xenograft model (Figures 6A and 6B, and Supplementary Figure 4A and 4B). Daily oral

administration of ASTX660 at 5, 10 and 20 mg/kg significantly inhibited tumor growth (P<0.05 from Day

15, 18 and Day 11, respectively) (Figure 6A). An intermittent schedule (cycles of 7 consecutive days of

dosing followed by 7 days of dose-holiday) at 20 mg/kg also achieved significant tumor growth inhibition

and its effects were equivalent to the continuous schedule in the same model (both P<0.05 vs vehicle

treatment from Day 8) (Figure 6B). The activity of ASTX660 was further investigated in an A375

melanoma xenograft model in nude mice (Figure 6C and Supplementary Figure 4C-i). Daily oral

administration of ASTX660 at 10 and 20 mg/kg caused significant tumor growth inhibition in an A375

model (both P<0.05 from Day 8), although in vitro this cell line was only sensitive to ASTX660 in the

presence of TNF- in vitro (Figure 4B and C).

In all three studies, the ASTX660 treatments were well-tolerated and no excessive bodyweight loss or

significant adverse effects was observed (Supplementary Figure 4A-i, B-i and C-i). It has been suggested




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that antagonizing multiple IAPs may be lead to reduced tolerability in vivo (20,36). To investigate the

inflammatory effects of ASTX660, we tested the compound ex vivo on peripheral blood mononuclear

cells (PBMCs) from 2 healthy human donors. We observed no substantial evidence of increase in

secretion of pro-inflammatory cytokines (TNF-α, IL-1β, IL-2, IL-6, IL-8, IFNγ and MCP-1) in a multiplexed

cytokine assay (Meso Scale Discovery) after a 48-hour treatment with 10 µM of ASTX660 or with 10 µM

LCL161 (see Supplementary Table 2). This was in contrast to the bivalent antagonist, BV-6 (14)

(Genentech), which causes rapid cIAP1/2 depletion, potently antagonises XIAP (37), and induced

cytokine elevation in the same PBMC assays (Supplementary Table 2).




15

Discussion

Antagonism of IAPs is considered a promising therapeutic approach for activating apoptosis pathways in

cancer and a number of IAP antagonists have undergone evaluation in early clinical trials (38). However,

despite several observations of objective clinical response, single agent activity in the clinic has been

limited (39). The N-terminal sequence (AVPI) of the endogenous protein SMAC provided the starting

point for the first generation of peptidomimetic IAP antagonists. The AVPI peptide shows cIAP1

selectivity (~200 fold over XIAP) due to differential interactions formed by the alanine residue in the P1

pocket of the BIR3 domains of XIAP and cIAP1. We have shown that this intrinsic selectivity is retained in

the first generation of SMAC mimetics currently in the clinic (27). Given its function in inhibiting caspase

activation, it has been proposed that XIAP antagonism plays a key role in the activation of apoptosis

through both the intrinsic and extrinsic pathways (2,40). An IAP antagonist with increased XIAP potency

may lead to improved efficacy due to more effective activation of apoptosis provided by releasing the

XIAP-block on caspases [38, 45]. An increased therapeutic window may also be achieved between

efficacious dose and the onset of cytokine-induced toxicity that results from cIAP antagonism. Cytokine

release syndrome has been reported as the dose-limiting toxicity in a Phase I dose escalation study of

cIAP selective antagonist LCL161 (23) and such effects have also been described in detail in preclinical

toxicity studies with another cIAP1/2 antagonist GDC-0152 (41). A true antagonist of cIAP1/2 and XIAP

could better explore the clinical potential of IAP antagonism.

In order to develop such a compound we have applied our fragment-based drug discovery approach

(PyramidTM) to specifically identify non-alanine leads with a cIAP1-cIAP2-XIAP profile, avoiding the

intrinsic selectivity resulting from peptidomimetic approaches based on the AVPI tetrapeptide (27,28). A

subsequent lead optimization campaign focused on reducing off-target activities and improving

pharmacokinetic properties, resulting in ASTX660, a potent non-peptidic IAP antagonist, structurally

distinct from IAP antagonists previously reported. The resulting IAP antagonism of ASTX660 was

demonstrated both in vitro and in vivo. ASTX660 binds to both cIAP1 and XIAP with low-nM potency and

this inhibition translates to cells, where potent cIAP1/2 antagonism was demonstrated by their

degradation, which occurs on the induction of autoubiquitinylation upon antagonist binding; at the

same time antagonism of XIAP was measured by displacement of SMAC and casapse-9. Moreover, our

data also confirm here that ASTX660 has potent long-lasting antagonism of cIAP1 and XIAP in vivo

following oral administration accompanied by significant inhibition of xenograft tumor growth. In the

assays used here ASTX660 demonstrated potencies of 2.8 and 0.22 nM, respectively, for XIAP and cIAP1




16

antagonism (12.7-fold difference). In contrast, other clinical stage monovalent SMAC mimetics do not

achieve this level of potency for XIAP (range 10-35 nM) or this balance of antagonism (39-227 fold

difference) under the same conditions (see Supplementary Table 1), making ASTX660 a novel compound

for further investigating the effects of IAP antagonism.

There are several potential advantages to antagonising XIAP in addition to cIAP1/2. Aberrantly high

levels of XIAP have been linked to poor prognosis in a number of tumor types, including diffuse large B-

cell lymphoma, renal cell carcinoma, bladder cancer and colorectal cancer (42-45), whilst overexpression

of XIAP in response to chemotherapy and radiotherapy has been proposed to contribute to resistance to

these treatments (42,46,47). In addition, XIAP levels have also been implicated in modulating responses

to immunotherapy, with increased levels in Hodgkin’s lymphoma preventing cytotoxic lymphocyte-

mediated cytotoxicity by blocking granzyme B-induced apoptosis (48) and upregulation of XIAP in

inflammatory breast cancer driving resistance to antibody-dependent cell-mediated cytotoxicity (49),

further indicating potential for a molecule that potently antagonizes XIAP. Furthermore, the role of XIAP

in both extrinsic and intrinisc apoptosis suggests that a potent XIAP inhibitor, such as ASTX660, might

also be expected to show increased synergy with activators of both these apoptotic pathways such as

chemotherapeutics, radiotherapy, or TRAIL agonists compared with a more cIAP selective antagonist (2).

It has been suggested that certain bivalent compounds that potently antagonise cIAP and XIAP are not

well tolerated in vivo as they induce an excessive cytokine-mediated pro-inflammatory phenotype

(20,36). However, we have demonstrated that, unlike the bivalent compounds, ASTX660 does not elicit a

toxic cytokine signature, suggesting that the IAP antagonist properties of this compound are not a

disadvantage in this respect.

ASTX660 is currently being tested in a phase 1-2 study in subjects with advanced solid tumors and

lymphomas (NCT02503423). Further studies with this novel compound will allow the true clinical

potential of an IAP antagonist to be explored both as a single agent and in combination.




17

Acknowledgements

We warmly acknowledge scientific discussions with many Astex colleagues.

We also thank Anne Cleasby for critical discussions and crystallographic support.




18

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21

Figure Legends

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 & 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, grey = neutral).

Hydrogen bonds between ligand and protein in C are shown as dashed red lines.

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 h 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 (upper panel). Total XIAP and SMAC levels were determined by

Western blots of cell lysates before immunoprecipitation (lower panel). C, A375 cell lysates were treated

with 1 µM ASTX660 for the indicated times. XIAP antagonism was measured by Western blotting for

SMAC levels after immunoprecipitation with anti-XIAP (upper panel). Total XIAP and SMAC levels were

determined by Western blots of cell lysates before immunoprecipitation (lower panel).

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 2h, lysed and relative levels of cIAP1 measured

using a Meso Scale Discovery plate-based assay. Results show the mean of duplicate values. B, A375

cells were treated with 1 µM ASTX660 for the indicated times and cIAP1 or XIAP levels analyzed by SDS-

PAGE followed by immunoblotting. -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 analysed by SDS-PAGE followed by immunoblotting. D, MDA-MB-231 cells were treated with the




22

indicated concentrations of ASTX660 for 2, 6, 24 and 48h, lysed and equal amounts of total protein were

then analysed by SDS-PAGE followed by immunoblotting with the indicated antibodies.

Figure 4

TNF- triggers ASTX660-induced apoptosis in cell lines. A, Viability (upper panel) and apoptosis (lower

panel) were measured in breast cancer MDA-MB-231 cells after treatment with 0.1 µM ASTX660 using

IncuCyte ZOOM live cell imaging. Anti-TNF- antibody at concentrations of 1 or 10 µg/mL was added to

neutralize TNF- in the cell supernatant in order to monitor effects on the cell survival. B, Melanoma

cell lines, A375 and SK-MEL-28, were treated with 1 µM ASTX660 plus or minus 1 ng/mL TNF- for 24 h.

Cells were then lysed, and equal amounts of total protein were analysed by SDS-PAGE followed by

immunoblotting with the indicated antibodies. C, A375 or SK-MEL-28 cells were treated with 1 µM

ASTX660 for 25 h in the presence or absence of 1 ng/mL TNF-. Cleaved caspase-3 activity was

measured by cytometry. D, Effect of 72 h ASTX660 treatment on the viability of 33 melanoma cell lines

(plus normal human dermal fibroblast (NHDF) cells (*), included as a control). Data generated by

CellTiterGlo in triplicate, in the presence or absence of 1 ng/mL TNF- were normalised 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).

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 analysed. 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.




23

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=7) and another was given 2 cycles of 7 consecutive days of dosing followed

by 7 days of dose-holiday (q.d. x 7/ no dose x 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. x 7/ no dose x 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.




Figure 1

P1

P2

P3

P4

Glu314

Asp309

Trp310

Trp323

Thr308

Tyr324

A B C




Figure 2

A B

C 0 5 10 15 20 25 30 40 50 60 120 240 Time (min)

XIAP

b-actin

SMAC

XIAP

SMAC

Cell Lysates

IP Anti-XIAP

0 5 10 15 20 25 30 40 50 60 120 240 Time (min)

IP Anti-XIAP

CellLysates

XIAP

SMAC

XIAP

SMAC

b-actin

0 0.001 0.01 0.1 1 10 [ASTX660], µM

0 0.001 0.01 0.1 1 10 [ASTX660], µM




A

C

Figure 3

B

D

0 5 10 15 20 25 30 40 50 60 120 240 Time (min)

cIAP1

XIAP

b-actin

cIAP2

cIAP1

b-actin

10 1 0.1 0.01 0.001 0 10 1 0.1 0.01 0.001 0 10 1 0.1 0.01 0.001 0 [ASTX660], µM

1 h 4 h 24 h

NIK

NF-kB2

p100

p52

cIAP1

cleaved PARP

cleaved caspase-3

phospho-IkBa[Ser32]

XIAP

b-actin

DMSO 0.001 0.01 0.1 1 10 DMSO 0.001 0.01 0.1 1 10 DMSO 0.001 0.01 0.1 1 10 DMSO 0.001 0.01 0.1 1 10 [ASTX660], µM2 h 6 h 24 h 48 h

phospho-p65[Ser536]

NF-kB1

p105

p50

total IkBa




A B

Figure 4

D

A375

DMSO: + - - - + - - -

TNF-a: - + - + - + - +

ASTX660: - - + + - - + +

SK-MEL-28

CleavedCaspase-9

CleavedCaspase-3

cIAP1

cIAP2

XIAP

Cleaved PARP

b-Actin

0

10

20

30

40

50

60

-4 -3 -2 -1 0 1 2

% C

ell

sC

leav

ed

Cas

pas

e-3

Po

siti

ve

Log 10 [ASTX660] (µM)

Control+ 1 ng/ml TNF-α

0

10

20

30

40

50

60

-4 -3 -2 -1 0 1 2

% C

ell

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leav

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pas

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Po

siti

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Log 10 [ASTX660] (µM)

Control

+ 1 ng/ml TNF-α

C

0

1

2

3

4

5

6

7

8

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01

D

Hs2

94

T

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05

8

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SK-M

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8

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75

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4

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95

T

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52

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-11

5

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61

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SK-M

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Figure 7: PD – MDA-MB-231 xenografts A

B D

C

Figure 5

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10

100

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10000

0 24 48 72 96 120 144 168

Me

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STX

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0 C

on

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Published OnlineFirst April 25, 2018.Mol Cancer Ther George A. Ward, Edward J Lewis, Jong Sook Ahn, et al. cancer cell lines and inhibits tumor growth

dependent apoptosis inαand XIAP, potently induces TNF-ASTX660, a novel non-peptidomimetic antagonist of cIAP1/2

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ASTX660, a novel non-peptidomimetic antagonist of cIAP1/2 ... · by fragment-based screening and...

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