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Cancer Therapy: Preclinical
The New Antitumor Drug ABTL0812 Inhibits theAkt/mTORC1 Axis by
Upregulating Tribbles-3PseudokinaseTatiana Erazo1, Mar Lorente2,3,
Anna L�opez-Plana4, Pau Mu~noz-Guardiola1,5,Patricia
Fern�andez-Nogueira4, Jos�e A. García-Martínez5, Paloma
Bragado4,Gemma Fuster4, María Salazar2, Jordi Espadaler5, Javier
Hern�andez-Losa6,Jose Ramon Bayascas7, Marc Cortal5, Laura Vidal8,
Pedro Gasc�on4,8,Mariana G�omez-Ferreria5, Jos�e Alf�on5, Guillermo
Velasco2,3, Carles Dom�enech5, andJose M. Lizcano1
Abstract
Purpose: ABTL0812 is a novel first-in-class, small moleculewhich
showed antiproliferative effect on tumor cells in pheno-typic
assays. Here we describe the mechanism of action of thisantitumor
drug, which is currently in clinical development.
Experimental Design:We investigated the effect of ABTL0812on
cancer cell death, proliferation, and modulation of intracel-lular
signaling pathways, using human lung (A549) and pancre-atic
(MiaPaCa-2) cancer cells and tumor xenografts. To identifycellular
targets, we performed in silico high-throughput screeningcomparing
ABTL0812 chemical structure against ChEMBL15database.
Results: ABTL0812 inhibited Akt/mTORC1 axis, resulting
inimpaired cancer cell proliferation and autophagy-mediated
celldeath. In silico screening led us to identify PPARs, PPARa
andPPARg as the cellular targets of ABTL0812. We showed
thatABTL0812 activates both PPAR receptors, resulting in
upregula-
tion of Tribbles-3 pseudokinase (TRIB3) gene expression.
Upre-gulated TRIB3 binds cellular Akt, preventing its activation
byupstream kinases, resulting in Akt inhibition and suppression
ofthe Akt/mTORC1 axis. Pharmacologic inhibition of PPARa/g orTRIB3
silencing prevented ABTL0812-induced cell death.ABTL0812 treatment
inducedAkt inhibition in cancer cells, tumorxenografts, and
peripheral bloodmononuclear cells frompatientsenrolled in phase
I/Ib first-in-human clinical trial.
Conclusions:ABTL0812has a unique and novelmechanismofaction,
that defines a new and drugable cellular route that linksPPARs to
Akt/mTORC1 axis, where TRIB3 pseudokinase plays acentral role.
Activation of this route (PPARa/g-TRIB3-Akt-mTORC1) leads to
autophagy-mediated cancer cell death. Giventhe low toxicity and
high tolerability of ABTL0812, our resultssupport further
development of ABTL0812 as a promising anti-cancer therapy. Clin
Cancer Res; 22(10); 2508–19. �2015 AACR.
IntroductionThe PI3K/AKT/mTORC1 (mTOR complex-1) signaling
path-
way drives signals from ligand-stimulated receptor
tyrosinekinases (RTK) to proteins that control cell metabolism,
growth,size, survival, and angiogenesis (1). In response to growth
orsurvival factors, phosphorylated RTKs activate PI3K, which
gen-erates the phosphatidylinositol 3,4,5-trisphosphate (PIP3)
sec-ondmessenger and allows the recruitment of protein kinaseAkt
tothe cellular membrane. Akt binds PIP3 through its PH
domain,resulting in a conformational change that enables its
activationthrough phosphorylation of two critical residues: Thr308
at theT-loop of the kinase domain by the
phosphoinositide-dependentkinase-1 (PDK1), and Ser473 at the
C-terminal hydrophobicmotif by mTOR complex-2 (mTORC2; refs. 2, 3).
Active Aktphosphorylates a plethora of substrates that regulate
cell survivaland metabolism, and also cell growth through
subsequent acti-vation of mTORC1. Akt phosphorylation of PRAS40 and
TSC2leads to activation of mTORC1, which promotes protein
trans-lation and synthesis by phosphorylating ribosomal
S6-Kinase(S6K) and the elongation factor 4EBP-1 (4).
Human cancer is very often associated with activation of
thePI3K/Akt/mTORC1 pathway, mainly due to overexpression or
1Protein Kinases and Signal Transduction Laboratory, Institut de
Neu-roci�encies and Departament de Bioquímica i Biologia Molecular,
Uni-versitat Aut�onoma de Barcelona, Bellaterra, Barcelona,
Spain.2Department of Biochemistry and Molecular Biology, Faculty
ofBiology, Universidad Complutense, Madrid, Spain. 3Instituto
deInvestigaci�on Sanitaria del Hospital Clínico San Carlos
(IdISSC),Madrid, Spain. 4Area of Molecular and Translational
Oncology,IDIBAPS, Fundaci�o Clínic, Universitat de Barcelona,
Barcelona, Spain.5Ability Pharmaceuticals, SL, Edifici Eureka,
Campus UAB, Bellaterra,Barcelona, Catalonia, Spain. 6Pathology
Department, Hospital Uni-versitari Vall d'Hebron, Barcelona, Spain.
7Institut de Neuroci�enciesand Department of Biochemistry and
Molecular Biology, UniversitatAut�onoma de Barcelona, Barcelona,
Spain. 8Medical OncologyDepartment, Novel Therapeutics Unit,
Hospital Clínic Barcelona.Barcelona, Catalonia, Spain.
Note: Supplementary data for this article are available at
Clinical CancerResearch Online
(http://clincancerres.aacrjournals.org/).
Corresponding Author: Jose M. Lizcano, Institut de
Neuroci�encies and Departa-ment de Bioquímica i Biologia Molecular,
Facultat de Medicina, UniversitatAut�onoma de Barcelona,
Bellaterra, Barcelona 08193, Spain. Phone: 34 935-813-076; Fax: 34
935-811-573; E-mail: [email protected]
doi: 10.1158/1078-0432.CCR-15-1808
�2015 American Association for Cancer Research.
ClinicalCancerResearch
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activatingmutations of RTKs and PI3K, to deletions/mutations
ofthe PIP3 phosphatase PTEN and to amplification of Akt (3,
5).These types of tumors are hypersensitive to inhibition of each
ofthe components of the PI3K/Akt/mTORC1 axis, and thereforemajor
efforts have been taken to develop inhibitors of thissignaling
pathway (6). Akt acts as a central node of this pathway,and
hyperactivated Akt is a common feature observed in humansolid
tumors (3, 5). Therefore, Akt has been proposed as aninteresting
target in cancer therapy. Two classes of Akt inhibitorsare now in
clinical development: ATP-competitive inhibitors(such as GSK690693)
which target active Akt, and allostericinhibitors (such as MK-2206
and perifosine, currently in phaseII and phase III clinical trials,
respectively) that by binding the PHdomain of Akt prevent its
activation by upstream kinases (3, 7).
Here we present ABTL0812, a first-in-class molecule with
anti-tumor activity which is currently in phase I/Ib clinical
trials inpatients with advanced solid tumors. We provide evidences
show-ing that ABTL0812 inhibits Akt phosphorylation by
upregulatingthe expressionofTRIB3,apseudokinase thatbindsand
inhibitsAkt,leading tomTORC1 inhibitionandautophagy-mediated cell
death.
MethodsCell culture, viability assay, transfection, and
lysis
Human cancer cell lines were purchased from ATCC
(2008;ATCC-authentication by isoenzymes analysis). Atg5þ/þ
andAtg5�/� T-large antigen–transformed MEFs were donated by
Dr.Mizushima (Tokyo Medical University, Tokyo, Japan). TRB3þ/þ
and TRB3�/� RasV12/E1A–transformed Mouse embryonic fibro-blasts
(MEF) have been described before (8). Cell lines werecultured as
recommended and transfected using Lipofectamine-2000.Cell
viabilitywas determinedby theMTT (3-(4, 5-dimethyl-2-thiazolyl)-2,
5-diphenyl-2H-tetrazolium Blue) reduction assay(9). Cells were
lysed in ice-cold RIPA buffer supplemented with 1mmol/L
sodium-orthovanadate, 50 mmol/L NaF, and 5
mmol/Lsodium-pyrophosphate, sonicated and stored at �20�C.
Immunoblotting and immunoprecipitationImmunoblot analyses were
performed following standard
procedures as described (10) and antibodies listed in the
Sup-
plementary Table S1. Akt was immunoprecipitated following
theprotocol previously described (11).
Transmission electron microscopyCells treated with ABTL0812 or
vehicle (ethanol) were pro-
cessed as described before (12). Pellets were embedded in
Epo-nate-12TM resin (Ted Pella). Ultrathin (70-nm thick)
sectionswere contrastedwith uranyl-acetate and lead citrate
solutions, andvisualized in transmission electron microscope (Jeol
JEM-1400)equipped with a CCD- GATAN-ES1000W Erlangshen camera.
Xenograft modelsFor A549 model, athymic female nude mice (n ¼ 5
per group)
were injected subcutaneously with 5 � 106 cells in each
flank.When tumors reached 80 to 100 mm3, mice were
randomlydistributed into treatment groups and administered the
corre-sponding treatments. ABTL0812 was administered by oral
gavageat 120 mg/kg every day. Docetaxel was administered by
intraper-itoneal route at 15 mg/kg once a week. MiaPaCa-2 cells
wereinjected in athymic male mice (n ¼ 5 per group) with 10 �
106cells in one flank. When tumors reached 80 to 160 mm3, micewere
randomly distributed and treated with vehicle or ABTL0812120 mg/kg
by oral gavage five times per week. Tumor volumeswere measured as
(length � width2)/2, twice a week. All proce-dures involving animal
were performed with the approval of theHospital Clinic Animal
Experimentation Committee, accordingto Spanish official
regulations.
Pharmacokinetics of ABTL0812 in mice and ratsABTL0812 was orally
(gavage, 100 mg/kg) and intravenously
(10 mg/kg) administered to male CD-1 mice. ABTL0812
wasquantified in plasma by validated LC/MS-MS methods and
phar-macokinetic parameters were calculated with
WinNonlinsoftware.
Clinical trial and isolation of PBMC from patientsABTL0812 is
currently in clinical evaluation in a phase I/Ib trial
in patients with advanced solid tumors. This study was
approvedby the local Ethics Committee of the Hospital Clinic
Barcelonaand the Spanish Agency of Medicines. The trial is
registered atwww.clinicaltrials.gov (NCT02201823). ABTL0812 was
admin-istered daily by the oral route at a dose of 1,000mg twice
daily fora period of 28 days. Blood samples for biomarker study
wereobtained by venipuncture before and after ABTL0812
treatment.PBMC were isolated by Ficoll (GE-Healthcare) density
centrifu-gation, lysed in RIPA buffer, sonicated, and stored at
�20�C.
Statistical analysisAll in vitro data were assessed using
one-way ANOVA followed
by Bonferroni multiple comparison test. Tumor volumes of
micewere compared using the ANOVA followed by t test.
Statisticalsignificance between the groups was assessed with the
log-ranktest (GraphPad). Levels of statistical significance were
set atP � 0.05.
ResultsABTL0812 has anticancer activity in vitro and in vivo
A novel class of chemically modified lipid-derived small
mole-cules was selected on the basis of two phenotypic assays:
anti-proliferative effect on tumor cells and low toxicity after
high doses
Translational Relevance
Hyperactivation of the Akt/mTORC1 axis is commonlyobserved in
many human cancers and blocking this pathwayis an important
anticancer strategy. Inhibition of Akt is a validtarget for
anticancer therapy and several AKT inhibitors areunder clinical
development. We present ABTL0812, a first-in-class small molecule
with a unique mechanism of action. Incells and tumor xenografts
models, ABTL0812 induces upre-gulation of TRIB3 pseudokinase. TRIB3
binds Akt, preventingits activation by upstream kinases and
resulting in an effectiveinhibition of the Akt/mTORC1 axis and in
autophagy-medi-ated cancer cell death. ABTL0812 is currently in
phase I/Ib first-in-human clinical trial in patients with advance
solid tumors.Preliminary results show that ABTL0812 also induces
Aktinhibition in humans. These evidences, together with a verylow
toxicity and high tolerability, support further develop-ment of
ABTL0812.
Inhibition of Akt/mTORC1 Axis by TRIB3 Pseudokinase
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Figure 1.ABTL0812 induces cancer cell death in vitro and reduces
tumor growth in human lung andpancreatic xenografts. A, chemical
structure of ABTL0812. B, ABTL0812 cellviability assays in human
cancer cell lines. IC50 values were obtained in MTT assays after
exposure to ABTL0812 for 48 hours. Values are the mean � SDof three
separate determinations. (Continued on the following page.)
Erazo et al.
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administration in rodents. From this class, ABTL0812was
selectedfor further preclinical development. The formula for
ABTL0812 is2-hydroxylinoleic acid (Fig. 1A).
ABTL0812 reduced viability of different human cancer cell
linestestedwith IC50 values of 20 to 60 mmol/L (Fig. 1B). Real-time
cellanalyses further demonstrated the antiproliferative effect
ofABTL0812 (Supplementary Fig. S1). ABTL0812 also induced
celldeath, which was confirmed by propidium iodide staining
(Sup-plementary Fig. S2). Importantly, ABTL0812 induced death
incancer cells but not in normal cells. Compared with
LN-18glioblastoma cells, primary astrocytes were resistant to
ABTL0812treatment at the highest ABTL0812 concentration tested
(200mmol/L; Fig. 1C).
To address ABTL0812 pharmacokinetics, we administered
thecompound to mice by oral and intravenous routes. ABTL0812showed
an excellent bioavailability by oral route (F:103%),with rapid oral
absorption (tmax:30 minutes), high pick concen-trations (Cmax:2.0
mg/mL), and wide volume of distribution(Vss:10.4 L/kg). Clearance
was also high (Cl: 82 mL/minute/kg).Overall, the compound was well
tolerated and animals appearedhealthy with no clinical signs of
distress, local or systemic toxicity.
To test the efficacy of ABTL0812 in inhibiting tumor growth
invivo, we used human lung and pancreatic xenograft models.
A549human lung cancer xenografts of nude mice were treated
withABTL0812 or the standard-of-care docetaxel. ABTL0812
inhibitedtumor progression with an efficacy similar to docetaxel
(Fig. 1D).Moreover, ABTL0812 lacked the toxic effects observed for
doc-etaxel, as shown by body weight measurements.
Similarly,ABTL0812 also inhibited the growth of MiaPaCa-2 human
pan-creatic cancer xenografts in nude mice, without affecting
bodyweight (Fig. 1E).
ABTL0812 induces autophagy-mediated cell deathNext, we
investigated the type of cell death induced by
ABTL0812 in A549 and MiaPaCa-2 cells. ABTL0812 did notinduce
nuclear fragmentation/condensation, or caspase-3 activa-tion
(Supplementary Fig. S3). Treatment with staurosporineresulted in
typical features of apoptosis, showing that A549 andMiaPaCa-2 cells
are apoptosis competent. These results indicatethat ABTL0812 fails
to induce apoptosis. In turn, several evidencesindicated that
ABTL0812-induced cell death is mediated byautophagy.
Two of the hallmarks of autophagy are the conversion of
thesoluble form of LC3 to a lipidated form associated to
autophago-somes (LC3-II), and the elimination of autophagosome
cargoproteins such as p62 (13). In A549 and MiaPaCa-2
cells,ABTL0812 induced the appearance of LC3-II and a decrease
onp62 protein in a concentration-dependent manner (Fig. 2A). Wealso
observed the occurrence of LC3-positive dots in cells treatedwith
ABTL0812, which indicates association to
autophagosomes(Supplementary Fig. S4). Electron microscopy analysis
ofABTL0812-treated A549 and MiaPaCa-2 cells revealed the pres-ence
of double membrane vacuolar structures with the morpho-logic
features of autophagosomes (Fig. 2B). Moreover, ABTL0812
also induced hallmarks of autophagy in vivo, as tumors
fromA549xenograftmice treatedwithABTL0812 showed an increase in
LC3-II levels, compared with mice treated with vehicle (Fig.
2C).
Next, we investigated the role of autophagy in the cell
deathinduced by ABTL0812. Pharmacologic inhibition of autophagywas
achieved by treating cells with a combination of the lyso-somal
protease inhibitors E64d and Pepstatin-A (PA) that blockthe final
step of autolysosomal degradation. Treatment of cellswith E64dþPA
prevented ABTL0812-induced cell death andresulted in an enhancement
of LC3-II accumulation in cellstreated with ABTL0812 (Fig. 2D),
indicating that ABTL0812induces dynamic autophagy in cancer cells.
Finally, we evaluatedthe activity of ABTL0812 in
oncogene-transformed embryonicfibroblasts derived from Atg5�/�
(autophagy-deficient) mice.Atg5 is an essential protein for
autophagosome formation(14). Transformed Atg5�/� MEF cells were
more resistant toABTL0812-induced cell death than transformed
Atg5þ/þ MEFcells, and did not activate autophagy in response to
ABTL0812treatment (Fig. 2E). Taken together, these results show
thatautophagy mediates ABTL0812-induced cancer cell death.
ABTL0812 inhibits the Akt/mTOR axisAutophagy induction is often
consequential to the inhibition
of mTOR signaling, as mTORC1 acts as a central regulator
inautophagy induction (15). We therefore investigated
whetherABTL0812-induced autophagy and cancer cell death occurred
viamTORC1 inhibition, by measuring levels of activation of
com-ponents of the Akt/mTORC1 signaling pathway. A549 and
Mia-PaCa-2 cells showed measurable phosphorylation levels for
allthe proteins of Akt/mTORC1 axis in basal conditions.
ABTL0812treatment reduced phosphorylation of ribosomal-S6 kinase
andof ribosomal-S6 protein (a well-established S6K substrate) in
aconcentration-dependent manner (Fig. 3).
Protein kinase Akt modulates the activation of mTORC1
byphosphorylating and inactivating the TSC2 and PRAS40 proteinsthat
act as repressors ofmTORC1 activity (1). Thus, Akt
inhibitionresults in impairedmTORC1 activity and activation of
autophagy.ABTL0812 induced Akt inhibition in A549 andMiaPaCa-2
cells ina dose-dependent manner, by reducing phosphorylation of
thetwo critical residues involved in Akt activation, Thr308
andSer473. Consequently, ABTL0812 also reduced phosphorylationof
theAkt substrates PRAS40 andTSC2 (Fig. 3). These results showthat
ABTL0812 induced inhibition of the Akt/mTORC1 axis incancer
cells.
ABTL0812 binds and induces PPARa and PPARgtranscriptional
activities, which mediate in ABTL0812-inducedcancer cell death
To identify themolecular targets of ABTL0812, we performed
insilico high-throughput screening comparing ABTL0812
chemicalstructure against the ChEMBL15 database
(https://www.ebi.ac.uk/chembldb/), following the similar property
and active analogprinciples (16). This approach allowed the
identification ofseveral ABTL0812 target candidates, including
PPARa andPPARg .
(Continued.) C, LN-18 glioblastoma cells or primary rat
astrocytes were incubated with ABTL0812, and 48 hours later cell
viability was monitored by MTTassays. Similar results were obtained
in three separate determinations. D and E, ABTL0812 inhibits tumor
growth in lung (D) and pancreatic (E) cancer xenograftmodels.
Athymic nude mice injected with A549 or MiaPaCa-2 cells were
treated with vehicle or ABTL0812 by oral gavage. Docetaxel was
administered byintraperitoneal injection. Tumor growth and weight
variation curves are shown. Results are the mean � SD of five mice
in each group. Because of toxicityin the docetaxel treatment, only
two mice were left at day 25 (n ¼ 2) and one after day 28 (n ¼ 1).
� , P < 0.05; �� , P < 0.01.
Inhibition of Akt/mTORC1 Axis by TRIB3 Pseudokinase
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Figure 2.ABTL0812 induces autophagy in vitro and in vivo.
Autophagy mediates ABTL0812-induced cancer cell death. A, cells
were treated with 50 to 200 mmol/LABTL0812 for 12 hours, lysed and
levels of the autophagy-marker proteins LC3 and p62 visualized by
immunoblotting. B, representative electron
microscopymicrophotographs showing autophagosomes (right) in cells
treated with 50 mmol/L ABTL0812 for 10 hours. C, total protein
extracted from A549 xenografttumors (Fig. 1C) were analyzed by
immunoblotting for expression of LC3. Figure shows a representative
analysis from vehicle (n ¼ 4) and treated (n ¼ 5)tumors. LC3-II
levels were normalized with actin and estimated in densitometric
units. D, A549 (white columns) or MiaPaCa-2 (black columns) cells
werepreincubated with vehicle or a combination of lysosomal
protease inhibitors E64d (10 mmol/L) and Pepstatin-A (PA, 10
mg/mL), before treatment with50 mmol/L ABTL0812 for 24 hours. Cell
viability was determined by MTT assay. Each value is the mean � SD
of three different experiments. ���P < 0.001 fromABTL0812
treated cells. Cells were lysed and LC3 lipidationwas visualized by
immunoblotting. E, Atg5þ/þ and Atg5�/�MEFswere treatedwith vehicle
or ABTL0812and cell viability determined 24 hours later. Values are
the mean � SD of three different experiments. � , P < 0.05; ���
, P < 0.001 from Atg5þ/þ MEFs.
Erazo et al.
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Of note, a possible interaction with PPARb/d was not
found.Putative ABTL0812-bindingmolecules identified in this
screeningwere further investigated through docking analysis. Figure
4Ashows themolecular model of ABTL0812 into the
ligand-bindingpocket of PPARg . In this model, ABTL0812 may bind
the PPARgligand-binding domain (LBD) through its carboxylate
group,which forms hydrogen bonds with the side of key amino
acidresidues (Ser289, His323, Tyr473, and His449), as it has
beenshown for other PPARg agonists such as rosiglitazone (17).
The2-hydroxyl group of ABTL0812 may also form hydrogen bondswith
residues Cys285 and Ser289, which are also conserved in thePPARa
LBD. This molecular model proposes that ABTL0812might establish
interactions similar to the specific PPARg agonistrosiglitazone,
thus predicting that ABTL0812 could act as PPARagonist. Indeed, in
vitro radioligand displacement assays usingpurifiedproteins showed
that ABTL0812binds PPARa andPPARgligand-binding pockets with Ki
values of 7.1 mmol/L and 4.7mmol/L, respectively (Supplementary
Fig. S5).
Next, we investigated the functional relevance of this
interac-tion, performing luciferase-based gene reporter assays in
cellstransfected with a PPAR-response element (PPRE) fused to
lucif-erase construct. In A549 and MiaPaCa-2, ABTL0812
inducedapproximately 5-fold PPAR transcriptional activity,
similarly tothe specific agonistsWY-14643 (PPARa) or rosiglitazone
(PPARg ;Supplementary Fig. S6). To determine whether
ABTL0812-induced PPAR activation was mediated by the PPAR isoformsa
and/or g , we performed analogous gene reporter assays in
cellstransiently transfected with plasmids encoding each
isoform.ABTL0812-induced PPAR transcriptional activity was higher
incells overexpressing PPARa or PPARg proteins, compared withthose
having the endogenous proteins only (Fig. 4B), suggestingthat
ABTL0812 functionally interacts with both receptors in cells.
Finally, we performed cell viability assays in the presence
ofPPARa/g antagonists. In A549 and MiaPaCa-2 cells, preincuba-tion
with PPARa antagonist GW6471 or PPARg antagonistGW9662 prevented
cell death induced by ABTL0812 (Fig. 4C),indicating that PPAR
receptorsmediate in ABTL0812-induced celldeath.
ABTL0812 activates PPARa- and PPARg-dependent expressionof
TRIB3
Koo and colleagues showed that PPARs regulate the expressionof
the pseudokinase Tribbles 3 (TRIB3), through binding a
PPAR-responsive element sequence within the TRIB3 promoter
(18).Other authors have shown that TRIB3 can interact with
Akt,preventing its activation by upstream kinases and resulting in
aninactive form of Akt (8, 19). Thus, we hypothesized thatABTL0812
could inhibit Akt (and downstream mTORC1) byupregulating TRIB3 via
PPARa/g activation. In A549 and Mia-PaCa-2 cells, ABTL0812 induced
a quick and robust increase inTRIB3 protein levels after 12 hours
of treatment (Fig. 5A), thatcorrelated with an increase in gene
transcription (Fig. 5B) andqPCR-measured TRIB3 mRNA levels (Fig.
5C). Although wecannot discard an effect of ABTL0812 on TRIB3
stability, theseresults indicate that ABTL0812 upregulates TRIB3
gene transcrip-tion. Furthermore, treatment with PPARa and/or PPARg
inhibi-tors GW6471 and GW9662, respectively, completely
abolishedthe ABTL0812-induced upregulation of TRIB3 gene
expression(Fig. 5C). Interestingly, treatment with either PPARa or
PPARgagonists resulted in increased TRIB3 expression and reduced
Aktphosphorylation (Fig. 5D). However, it was necessary to
combinetreatment with both agonists to obtain similar levels of
inhibitionof Akt (phospho-PRAS40) andmTORC1 activities
(phospho-S6Kand phospho-S6) than those induced by ABTL0812.
Altogether,our results demonstrate that ABTL0812 induces TRIB3
geneexpression via activation of PPARa and PPARg activities.
ABTL0812 inhibits Akt and mTORC1 via TRIB3To investigate the
role of upregulated TRIB3 on Akt activation,
we overexpressed increasing amounts of TRIB3 in A549
andMiaPaCa-2 cells. We observed reduced phosphorylation of Aktand
Akt substrate PRAS40 that inversely correlated with TRIB3expression
(Fig. 6A). This was probably due to a TRIB3–Aktphysical
interaction, because ABTL0812 treatment increased theamount of Akt
coimmunoprecipitated with TRIB3 (Fig. 6B).Moreover, unlike
wild-type transformed MEF, TRIB3-deficientMEF cells did not show
Akt (Ser473) or mTORC1 (pS6) inhibi-tion, or induction of autophagy
(LC3-II marker) in response toABTL0812 treatment (Fig. 6C). Cell
viability assays showed thatTRIB3-deficient MEFs are resistant to
ABTL0812-induced celldeath, unlike wild-type transformedMEFs (Fig.
6D). These resultssupport a pivotal role of TRIB3 on the impairment
of the Akt/mTORC1 axis and on the autophagy-mediated cancer cell
deathobserved in response to ABTL0812 treatment.
ABTL0812 induces TRIB3 upregulation and Akt inhibitionin
vivo
To determine the in vivo relevance of our findings, we
nextinvestigated the effect of ABTL0812 in A549 human lung
andMiaPaCa-2 human pancreatic cancer xenografted mice (Fig. 1Cand
D). Immunohistochemical and immunoblotting analysesshowed that
ABTL0812-induced upregulation of TRIB3 protein
Figure 3.ABTL0812 inhibits the Akt/mTORC1 axis. A549 and
MiaPaCa-2 cells treatedwith vehicle (0) or ABTL0812 (mmol/L) for 24
hours were lysed, and levels ofphosphorylated proteins were
analyzed by immunoblotting. Levels of totalproteins and actinwere
used as control. Similar results were obtained in threeseparate
experiments.
Inhibition of Akt/mTORC1 Axis by TRIB3 Pseudokinase
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and inhibition of Akt phosphorylation (Ser473) in A549
andMiaPaCa-2 tumors (Fig. 6E). These data, together with that
pre-sented in Fig. 2 showing that ABTL0812 induces autophagy inA549
xenografts, suggest that the antitumor activity of ABTL0812in vivo
relies on inhibition of Akt and induction of autophagy-mediated
cell death.
Akt phosphorylation is a pharmacodynamic marker in
patientstreated with ABTL0812
Finally, we evaluated the activity of ABTL0812 in some
patientsincluded in phase I/Ib clinical trials (NCT02201823),
monitoringAkt phosphorylation levels in PBMCs. We collected blood
sam-ples, pretreatment and at different days of chronic treatment
with1,000 mg of ABTL0812, a dose that was safe and well
tolerated(preliminary data not shown). Our preliminary results show
that
treatment with ABTL0812 resulted in a marked reduction on
Aktphosphorylation in PBMCs from 3 patients (Fig. 6F). We couldnot
detect TRIB3 protein in PBMCs, using commercially
availableanti-TRIB3 antibodies. However, parallel qPCR analysis
showed asignificant increase in TRIB3 mRNA levels in human
PBMCstreated in vitro with ABTL0812 (Supplementary Fig. S7),
thussuggesting that ABTL0812 also induces Akt inhibition in
humansthrough upregulation of TRIB3.
DiscussionHere,wepresent themechanismof actionofABTL0812, a
novel
and first-in-class antitumor drug which is currently in phase
I/Ibclinical trials in patientswith advanced solid tumors. ABTL0812
isa polyunsaturated fatty acid derivative, small molecule that
Figure 4.ABTL0812 induces PPARa and PPARg transcriptional
activities, which mediate in ABTL0812-induced cancer cell death. A,
model for the binding of ABTL0812(depicted in yellow) to PPARg
ligand binding domain, showing the interactions (dotted green
lines) of the carboxylate and hydroxyl groups of ABTL0812(depicted
in red) with the side chains of residues (depicted in blue) within
PPARg active site. B, luciferase-PPRE reporter and pRL-CMV-Renilla
plasmidswere cotransfected with plasmids encoding empty backbone or
PPARa or PPARg . Sixteen hours posttransfection, cells were treated
with vehicle or50 mmol/L ABTL0812. Twenty-four hours later, lysates
were subjected to dual-luciferase assay. Values are the mean � SD
of three different experiments,each performed in triplicate and
normalized using Renilla values. ���P < 0.001 from cells
transfected with empty plasmid. C, cells were preincubated
withvehicle (white columns), 0.2 mmol/L GW6471 (PPARa-antagonist)
or 1 mmol/L GW9662 (PPARg-antagonist) during 2 hours, and then
treated with50 mmol/L ABTL0812 for 24 hours. Cell viability was
measured by MTT analysis. Values are the mean � SD of three
different determinations. � , P < 0.05;��� , P < 0.001 from
ABTL0812 treated cell.
Erazo et al.
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induces cell death in a broad panel of cancer cell lines,
whereas itdoes not affect cell viability of nontumorogenic cells.
ABTL0812also inhibits tumor growth in human lung and pancreatic
tumorxenografts. Herein, we elucidated a novel cell signaling
pathwaythrough which ABTL0812 induces autophagic cell death.
Weshowed that ABTL0812 induces upregulation of the TRIB3
pseu-dokinase through activation of PPARa and PPARg
transcriptionfactors.Upregulated TRIB3binds toAkt andprevents its
activationby PDK1 and mTORC2 upstream kinases, resulting in
inhibitionof the Akt/mTORC1 axis and, therefore, autophagy-mediated
celldeath (Fig. 6G).
Autophagy is an essential process that consists of
selectivedegradation of cellular components, assuring the
maintenanceof cellular homeostasis via lysosomal degradation
pathways (13).The role of autophagy in cancer is still far from
clear, as it can act asa tumor suppressor and tumor promoter,
depending on tumor
type, stage, and genetic context, as well as on the duration
andstrengthof the triggering stimuli (15, 20).Our results
demonstratethat ABTL0812 induces autophagy-mediated cancer cell
death invitro and in vivo, without activating cellular apoptosis.
Theseresults are in agreementwith those reporting that
polyunsaturatedfatty acid and derivatives exert their
antiproliferative actionthrough activation of autophagy
(21–23).
The majority of current anticancer treatments activate
apopto-sis, and resistance to chemotherapy is a major challenge in
cancer(24). Autophagy-mediated cell death has emerged as an
alterna-tive to kill cancer cells without inducing resistance to
apoptosisinducer drugs (25). mTORC1 plays a central role in
regulatingcellular autophagy, and mTORC1 inhibitors induce
autophagy-mediated cell death in many systems, such as AZD8055
inhepatocellular carcinoma (26) or Ku0063794 and temsirolimusin
renal carcinoma (27). On the other hand, mTORC1 activation
Figure 5.ABTL0812 induces upregulation of the Akt endogenous
inhibitor TRIB3 through PPARa/g activation. A, cells treated with
ABTL0812 for the indicated timeswere lysed, and levels of TRIB3 and
actin analyzed by immunoblotting. B, cells were transfected with
luciferase-TRIB3 promoter reporter and pRL-CMV-Renillaplasmids.
Sixteen hours posttransfection, cells were treated with vehicle or
100 mmol/L ABTL0812. Twenty-four hours later, lysates were
subjected todual-luciferase assay. Values are the mean � SD of
three different experiments, each performed in triplicate and
normalized using Renilla values.C, cells were preincubated with
vehicle or PPARa antagonist GW6471 (0.2 mmol/L) and/or PPARg
antagonist GW9662 (1 mmol/L) for 2 hours, and treatedfor 24 hours
with vehicle or 50 mmol/L ABTL0812. TRIB3 mRNA levels were
monitored by RT-qPCR. Values are the mean � SD of three different
determinations,each performed in duplicate. ���P < 0.001 from
vehicle-treated cells. D, A549 cells were treated with PPARa
agonist WY14643 (1 mmol/L) and/or PPARg agonistrosiglitazone (1
mmol/L) for 24 hours, lysed and proteins analyzed by
immunoblotting.
Inhibition of Akt/mTORC1 Axis by TRIB3 Pseudokinase
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Figure 6.ABTL0812-evoked TRIB3 upregulation inhibits Akt
activity in vitro and in vivo. A, cells transfected with empty or
increasing amounts of a plasmid encodinghuman-TRIB3 were lysed and
levels of proteins monitored by immunoblotting. B,
immunoprecipitation of Akt from MiaPaCa-2 lysates after treatment
withvehicle or ABTL0812 for 24 hours. Top, levels of Akt and TRIB3
in the immunoprecipitates. C, wild-type and TRIB3-KO MEF cells were
treated with ABTL0812for 24 hours, and levels of the indicated
proteins were analyzed by immunoblotting. D, wild-type and TRIB3-KO
MEFs were treated with ABTL0812 for24 hours, and cell viability
measured by MTT analysis. Values are the mean � SD of three
different determinations. ���P < 0.001 from TRIB3 wt. E, A549
andMiaPaCa-2 tumors were collected after sacrifice, and TRIB3 was
evaluated by immunohistochemical analysis (left; scale bars: 100
mm). Levels ofphospho-Akt(Ser473) were evaluated in total protein
extracts, by immunoblotting (right). Histograms show the
corresponding quantifications.� , P < 0.01; �� , P < 0.005;
��� , P < 0.001 from vehicle-treated tumors. F, phospho-Akt and
Akt levels in PBMCs from patients before (day 0) or after1,000 mg
bid ABTL0812 oral chronic treatment. Protein extracts were analyzed
by immunoblotting for phospho-Akt-Ser473 and total Akt. Upper
graphsshow the quantification of levels of phospho-Akt normalized
with Akt protein, estimated by densitometric analysis (day 1 ¼
100%). G, a model showing themechanism of action of ABTL0812.
Erazo et al.
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is frequently associated with resistance to antitumor drugs (6).
AsABTL0812 is a potent inhibitor of the Akt/mTORC1 axis,
itsadministration in combination with standard
chemotherapeuticdrugs might prove effective in therapy-resistant or
apoptosis-refractory tumor. This has been shown for the mTORC1
inhibitoreverolimus (RAD001), which sensitizes papillary thyroid
cancercells to treatment with doxorubicin by an
autophagy-mediatedmechanism (28).
Here we present in vitro and in vivo evidences showing
thatABTL0812binds to and activates PPARa andPPARg
transcriptionalactivities, and that pharmacologic inhibition of
both receptorsprevents ABTL0812-induced cancer cell death,
suggesting thatPPARa/g are the primary molecular targets of
ABTL0812 in thecell. PPARs are nuclear receptors that
heterodimerize with theretinoid-X receptors to modulate gene
transcription. Mammaliancell expresses three PPAR isoforms, PPARa,
PPARb/d, and PPARg(29). Using an in silico analysis, we identified
PPARa and PPARg astargets of ABTL0812, but not PPARb/d. Several
authors have shownthat polyunsaturated and derivative long fatty
acids activate PPARstranscriptional activities (21, 30).
Interestingly, the docking experi-ments performed in our study show
that the a-hydroxyl group inABTL0812 forms two extra hydrogen bonds
with the critical resi-dues Cys285/Ser289 in PPARg LBD (also
conserved in the PPARaLBD), compared with the nonhydroxylated
linoleic acid. Accord-ingly, ABTL0812 showed higher PPARg agonistic
activity (Ki �5mmol/L) than linoleate (79 mmol/L) or the PPARg
natural agonistsflavonoids such as kaempferol (30 mmol/L; ref.
30).
PPARs regulate the expression of many genes involved inglucose
and lipid metabolism (29, 31). Interestingly, activationof PPARg
reduces cell proliferation and invasion, and enhancesapoptosis in
different cancer models such as breast and hepato-carcinoma (21,
32), whereas activation of PPARa inhibitstumor growth and
angiogenesis in mouse models (33–35).Therefore, activators of PPARa
or PPARg have been proposedas anticancer drugs, and specific
synthetic PPARa or PPARgagonists are in clinical trials for
treating cancer, such as the PPARgagonist efatutazone or the PPARa
agonist fenofibrate, in patientswith liposarcoma (NTC02249949) or
multiple myeloma(NCT01965834), respectively. ABTL0812, by
activating PPARaand PPARg , might well synergize the anticancer
activity of eachreceptor. Importantly, single treatment of the
cancer cells used inour study with either PPARa or PPARg agonist
induced partialinhibition of mTORC1 activity, whereas combined
treatmentwith both agonists resulted in a robust mTORC1
inhibition,similar to that obtained for ABTL0812 treatment (Fig.
5D). Giventhe fact that anewgenerationof dual PPARa/g agonists has
shownpromising anticancer activity (i.e., TZD18 inbreast cancer;
ref. 36),it will be of interest to investigatewhether
PPARa/gmechanismofaction also involves inhibition of the Akt/mTORC1
axis.
Our most striking result is the observation that upregulation
ofTribbles pseudokinase-3, through activation of PPARa/g
recep-tors, plays a crucial role in the anticancer activity of
ABTL0812.TRIB3 is a highly conserved protein which lacks critical
catalyticresidues for binding ATP and therefore lacks kinase
activity (37).Together with TRIB1 and TRIB2 proteins, TRIB3 define
the Trib-bles subfamily of pseudokinases, which was first described
inDrosophila as regulators of cell proliferation andmigration
duringdevelopment (38). As it happens for other pseudokinases,
TRIB3exerts its function in the cell interactingwith several
proteins, suchas the transcription factors SMAD3 (39) and ATF-4
(40), ormembers of the MAPK family (41). Of note, few authors
have
shown that TRIB3 can interact with Akt, preventing its
phosphor-ylation by upstream kinases and resulting in suppression
of theinsulin pathway (16, 19, 42). Here we show that
ABTL0812-evoked TRIB3 binds Akt, and that TRIB3 overexpression in
cellsresulted inAkt inhibition.Moreover, we also found that
treatmentwith ABTL0812 enhances the interaction between TRIB3 and
Akt,in line with previous data obtained in glioma cells and
oncogene-transformed MEFs, where a similar increase in TRIB3 levels
andTRIB3-Akt interaction occur upon exposure to
D9-tetrahydrocan-nabinol (THC; refs. 12). TRIB3 interacts more
strongly withinactive/dephosphorylated Akt than active Akt (M. S.,
unpub-lished results), therefore it is likely that overexpressed
TRIB3could trap the inactive form of Akt, preventing its
activation/phosphorylation by upstream kinases. Whether TRIB3 masks
thephosphorylatable residues in Akt (Thr308 and Ser473), induces
aconformational change that impairs recognition by upstreamkinases
PDK1 and mTORC2, or hampers the binding of the AktPHdomain to PIP3
remains to be clarified.On the other hand, it isalso likely that,
in addition to changes in TRIB3 protein levels,there could exist
additional mechanisms involved in regulatingthe interaction of
TRIB3 with its protein targets, and specificallywith Akt.
Posttranslational modifications of TRIB3, yet to bedescribed,
and/or changes in the interaction with other membersof the Tribbles
family that participate in modulating these inter-actions, are two
plausible possibilities that are currently investi-gated in our
laboratories.
Our results indicate that TRIB3 plays a central role in
themechanism of action of ABTL0812, as it has been proposed
forother antitumoral drugs, such as THC in glioma and
hepatocel-lular carcinoma tumors (43, 44) or salinomycin in lung
cancercells (45). Interestingly, these drugs also induce autophagy,
as itdoes ABTL0812, suggesting that TRIB3 upregulation
effectivelyprovokes a robust inhibition of the Akt/mTORC1 axis in
vivo. THCand salinomycin exert their action by stimulation of
endoplasmicreticulum stress and then activating ATF4/CHOP
transcriptionfactors that regulate TRIB3 gene. In our study, we
identify a newroute that links PPARa/g activation, inhibition of
Akt/mTORC1via TRIB3 and cancer cell death.
There are contradictory reports in the literature regarding the
roleof TRIB3 in human cancer. Some laboratories reported
elevatedTRIB3 mRNA levels which correlated with bad prognosis in
colo-rectal cancer (46) and breast cancer patients (47). On the
otherhand, high TRIB3 protein levels have been associated with
goodprognosis sensitivity to radiotherapy in breast cancer patients
(48).As TRIB3 is a very stable protein (49), the discrepancies
observedbetween TRIB3 mRNA and protein levels in human breast
cancerprognosis might be due to posttranslational modifications,
still tobe described, that might modulate the ability of TRIB3 to
interactwith other proteins that regulate tumor growth. In this
study,however, we show that ABTL0812 treatment induces a
robustincrease inTRIB3 transcriptionandmRNAandprotein
levels,whichcorrelate with cancer cell death and tumor growth
inhibition. Ourresults are in agreement with a recent report
showing that geneticinhibition of TRIB3 enhances tumorogenesis in
different cancermodels (8), and support a role for TRIB3 as a tumor
suppressor.
Hyperactivation of the Akt/mTORC1 axis is observed in
themajority of human cancers and blocking this pathway is
animportant anticancer strategy (5).Wehave shownhere the activityof
ABTL0812 in a panel of cancer cell lines that express high levelsof
phosphorylated/active Akt. Importantly, we have preliminaryobserved
activity of ABTL0812 in patients. ABTL0812 treatment
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impaired Akt phosphorylation in PBMCs from patients includedin
the ongoing phase I clinical trial (Fig. 6F). Therefore,
Aktphosphorylation will be used as a surrogated
pharmacodynamicbiomarker, for estimating the recommended dose for
the forth-coming phase II trials. We have preliminarily observed
increasedTRIB3 mRNA levels in human PBMCs treated with
ABTL0812(Supplementary Fig. S7), suggesting that TRIB3 qPCR
analysiscould be also used tomonitor the biologic activity of
ABTL0812 inhumans. Finally, and given its uniquemechanismof action,
it willbe important to evaluate ABTL0812 in combination with
stan-dard chemotherapeutical compounds as well as with other
targettherapies. We foresee ABTL0812-based therapies will
contributeto improve patients' treatment and quality of life.
Disclosure of Potential Conflicts of InterestJ. Espadaler has
ownership interest (including patents) in and is a consultant/
advisory board member for Ability Pharmaceuticals SL. J.M.
Lizcano is aconsultant/advisory board member for Ability
Pharmaceuticals. No potentialconflicts of interest were disclosed
by the other authors.
Authors' ContributionsConception and design: M. Salazar, J.
Espadaler, M. G�omez-Ferreria, J. Alf�on,C. Dom�enech, J.M.
LizcanoDevelopment of methodology: T. Erazo, M. Lorente, A.
L�opez-Plana,P. Mu~noz-Guardiola, P. Fern�andez-Nogueira, G.
Fuster, J. Hern�andez-Losa,J.M. LizcanoAcquisition of data
(provided animals, acquired and managed patients,provided
facilities, etc.): T. Erazo, M. Lorente, P. Mu~noz-Guardiola,P.
Fern�andez-Nogueira, J.A. García-Martínez, P. Bragado, M.
Salazar,J. Hern�andez-Losa, J.R. Bayascas, L. Vidal, G.
VelascoAnalysis and interpretation of data (e.g., statistical
analysis, biostatistics,computational analysis): T. Erazo, M.
Lorente, A. L�opez-Plana,P. Fern�andez-Nogueira, J.A.
García-Martínez, P. Bragado, M. Salazar,J. Espadaler, M. Cortal, J.
Alf�on, C. Dom�enech, J.M. Lizcano
Writing, review, and/or revision of the manuscript: M.
Lorente,A. L�opez-Plana, P. Fern�andez-Nogueira, G. Fuster, J.
Espadaler, J. Hern�andez-Losa, J.R. Bayascas, M. Cortal, L. Vidal,
P. Gasc�on, M. G�omez-Ferreria, J. Alf�on,G. Velasco, C. Dom�enech,
J.M. LizcanoAdministrative, technical, or material support (i.e.,
reporting or organizingdata, constructing databases): J.M.
LizcanoStudy supervision: P. Gasc�on, G. Velasco, J.M. Lizcano
AcknowledgmentsThe authors thank Pablo Escrib�a and Xavier
Busquets (Universitat Illes
Balears, Spain) for the preliminary identification of ABTL0812
as an anticanceragent (patent number WO/2010/106211) and Giovani
Cincilla, EmilianaD'Oria, and Oscar Villaca~nas (Intelligent Pharma
SL) for in silico analysis, LuisBotella and Javier Soto
(Medalchemy) for the synthesis of ABTL0812, andWashington
Biotechnology Inc. for preliminary preclinical studies. The
authorsalso thank Victor Yuste for helpful discussions, Cristina
Gutierrez for tissueculture assistance, and Servei de Gen�omica i
Inform�atica from the UAB.
Grant SupportThis work was supported by grants from the
Government of Catalonia
(ACCIO/FINEBT10-1-0047), and from the Spanish Ministry of
Economyand Competitiveness (MINECO), (CDTI/PID/IDI-20101630,
GenomaEspa~na/INNOCASH/2011196, ENISA/EBT2012/100375,
INNPACTO/IPT-2012-0614-010000). J. Alf�on and M. G�omez-Ferreria
were funded by Torres-Quevedo grant (MINECO), and M. Cortal by an
Inncorpora grant (MINECO).Work in G. Velasco's laboratory was
supported by grants from Spanish Ministryof Economy and
Competitiveness (MINECO), Fondo Europeo de desarrolloRegional
(FEDER) (PI12/02248 and PI15/00339) and Fundaci�on MutuaMadrile~na
(AP101042012).
The costs of publication of this articlewere defrayed inpart by
the payment ofpage charges. This article must therefore be hereby
marked advertisement inaccordance with 18 U.S.C. Section 1734
solely to indicate this fact.
Received July 30, 2015; revised November 20, 2015; accepted
November 30,2015; published OnlineFirst December 15, 2015.
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www.aacrjournals.org Clin Cancer Res; 22(10) May 15, 2016
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Inhibition of Akt/mTORC1 Axis by TRIB3 Pseudokinase
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Upregulating Tribbles-3 PseudokinaseThe New Antitumor Drug ABTL0812
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