The New Antitumor Drug ABTL0812 Inhibits the Akt/Mtorc1 Axis By

Published OnlineFirst December 15, 2015; DOI: 10.1158/1078-0432.CCR-15-1808

Cancer Therapy: Preclinical Clinical Cancer Research The New Antitumor Drug ABTL0812 Inhibits the Akt/mTORC1 Axis by Upregulating Tribbles-3 Pseudokinase Tatiana Erazo1, Mar Lorente2,3, Anna Lopez-Plana 4, Pau Munoz-Guardiola~ 1,5, Patricia Fernandez-Nogueira 4, Jose A. García-Martínez5, Paloma Bragado4, Gemma Fuster4, María Salazar2, Jordi Espadaler5, Javier Hernandez-Losa 6, Jose Ramon Bayascas7, Marc Cortal5, Laura Vidal8, Pedro Gascon 4,8, Mariana Gomez-Ferreria 5, Jose Alfon 5, Guillermo Velasco2,3, Carles Domenech 5, and Jose M. Lizcano1

Abstract

Purpose: ABTL0812 is a novel first-in-class, small molecule tion of Tribbles-3 pseudokinase (TRIB3) gene expression. Upre- which showed antiproliferative effect on tumor cells in pheno- gulated TRIB3 binds cellular Akt, preventing its activation by typic assays. Here we describe the mechanism of action of this upstream kinases, resulting in Akt inhibition and suppression of antitumor drug, which is currently in clinical development. the Akt/mTORC1 axis. Pharmacologic inhibition of PPARa/g or Experimental Design: We investigated the effect of ABTL0812 TRIB3 silencing prevented ABTL0812-induced cell death. on cancer cell death, proliferation, and modulation of intracel- ABTL0812 treatment induced Akt inhibition in cancer cells, tumor lular signaling pathways, using human lung (A549) and pancre- xenografts, and peripheral blood mononuclear cells from patients atic (MiaPaCa-2) cancer cells and tumor xenografts. To identify enrolled in phase I/Ib first-in-human clinical trial. cellular targets, we performed in silico high-throughput screening Conclusions: ABTL0812 has a unique and novel mechanism of comparing ABTL0812 chemical structure against ChEMBL15 action, that defines a new and drugable cellular route that links database. PPARs to Akt/mTORC1 axis, where TRIB3 pseudokinase plays a Results: ABTL0812 inhibited Akt/mTORC1 axis, resulting in central role. Activation of this route (PPARa/g-TRIB3-Akt- impaired cancer cell proliferation and autophagy-mediated cell mTORC1) leads to autophagy-mediated cancer cell death. Given death. In silico screening led us to identify PPARs, PPARa and the low toxicity and high tolerability of ABTL0812, our results PPARg as the cellular targets of ABTL0812. We showed that support further development of ABTL0812 as a promising anti- ABTL0812 activates both PPAR receptors, resulting in upregula- cancer therapy. Clin Cancer Res; 1–12. 2015 AACR.

Introduction The PI3K/AKT/mTORC1 (mTOR complex-1) signaling path- 1 Protein Kinases and Signal Transduction Laboratory, Institut de Neu- way drives signals from ligand-stimulated receptor tyrosine rociencies and Departament de Bioquímica i Biologia Molecular, Uni- versitat Autonoma de Barcelona, Bellaterra, Barcelona, Spain. kinases (RTK) to proteins that control cell metabolism, growth, 2Department of Biochemistry and Molecular Biology, Faculty of size, survival, and angiogenesis (1). In response to growth or Biology, Universidad Complutense, Madrid, Spain. 3Instituto de survival factors, phosphorylated RTKs activate PI3K, which gen- Investigacion Sanitaria del Hospital Clínico San Carlos (IdISSC), erates the phosphatidylinositol 3,4,5-trisphosphate (PIP3) sec- Madrid, Spain. 4Area of Molecular and Translational Oncology, IDIBAPS, Fundacio Clínic, Universitat de Barcelona, Barcelona, Spain. ond messenger and allows the recruitment of protein kinase Akt to 5Ability Pharmaceuticals, SL, Ediﬁci Eureka, Campus UAB, Bellaterra, the cellular membrane. Akt binds PIP3 through its PH domain, 6 Barcelona, Catalonia, Spain. Pathology Department, Hospital Uni- resulting in a conformational change that enables its activation versitari Vall d'Hebron, Barcelona, Spain. 7Institut de Neurociencies and Department of Biochemistry and Molecular Biology, Universitat through phosphorylation of two critical residues: Thr308 at the Autonoma de Barcelona, Barcelona, Spain. 8Medical Oncology T-loop of the kinase domain by the phosphoinositide-dependent Department, Novel Therapeutics Unit, Hospital Clínic Barcelona. kinase-1 (PDK1), and Ser473 at the C-terminal hydrophobic Barcelona, Catalonia, Spain. motif by mTOR complex-2 (mTORC2; refs. 2, 3). Active Akt Note: Supplementary data for this article are available at Clinical Cancer phosphorylates a plethora of substrates that regulate cell survival Research Online (http://clincancerres.aacrjournals.org/). and metabolism, and also cell growth through subsequent acti- Corresponding Author: Jose M. Lizcano, Institut de Neurociencies and Departa- vation of mTORC1. Akt phosphorylation of PRAS40 and TSC2 ment de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat leads to activation of mTORC1, which promotes protein trans- Autonoma de Barcelona, Bellaterra, Barcelona 08193, Spain. Phone: 34 935- lation and synthesis by phosphorylating ribosomal S6-Kinase 813-076; Fax: 34 935-811-573; E-mail: [email protected] (S6K) and the elongation factor 4EBP-1 (4). doi: 10.1158/1078-0432.CCR-15-1808 Human cancer is very often associated with activation of the 2015 American Association for Cancer Research. PI3K/Akt/mTORC1 pathway, mainly due to overexpression or

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plementary Table S1. Akt was immunoprecipitated following the Translational Relevance protocol previously described (11). Hyperactivation of the Akt/mTORC1 axis is commonly observed in many human cancers and blocking this pathway Transmission electron microscopy is an important anticancer strategy. Inhibition of Akt is a valid Cells treated with ABTL0812 or vehicle (ethanol) were pro- target for anticancer therapy and several AKT inhibitors are cessed as described before (12). Pellets were embedded in Epo- under clinical development. We present ABTL0812, a first-in- nate-12TM resin (Ted Pella). Ultrathin (70-nm thick) sections class small molecule with a unique mechanism of action. In were contrasted with uranyl-acetate and lead citrate solutions, and cells and tumor xenografts models, ABTL0812 induces upre- visualized in transmission electron microscope (Jeol JEM-1400) gulation of TRIB3 pseudokinase. TRIB3 binds Akt, preventing equipped with a CCD- GATAN-ES1000W Erlangshen camera. its activation by upstream kinases and resulting in an effective inhibition of the Akt/mTORC1 axis and in autophagy-medi- Xenograft models fi ated cancer cell death. ABTL0812 is currently in phase I/Ib rst- For A549 model, athymic female nude mice (n ¼ 5 per group) in-human clinical trial in patients with advance solid tumors. were injected subcutaneously with 5 106 cells in each flank. Preliminary results show that ABTL0812 also induces Akt When tumors reached 80 to 100 mm3, mice were randomly inhibition in humans. These evidences, together with a very distributed into treatment groups and administered the corre- low toxicity and high tolerability, support further develop- sponding treatments. ABTL0812 was administered by oral gavage ment of ABTL0812. at 120 mg/kg every day. Docetaxel was administered by intraperitoneal route at 15 mg/kg once a week. MiaPaCa-2 cells were injected in athymic male mice (n ¼ 5 per group) with 10 106 cells in one flank. When tumors reached 80 to 160 mm3, mice activating mutations of RTKs and PI3K, to deletions/mutations of were randomly distributed and treated with vehicle or ABTL0812 the PIP3 phosphatase PTEN and to amplification of Akt (3, 5). 120 mg/kg by oral gavage five times per week. Tumor volumes 2 These types of tumors are hypersensitive to inhibition of each of were measured as (length width )/2, twice a week. All proce- the components of the PI3K/Akt/mTORC1 axis, and therefore dures involving animal were performed with the approval of the major efforts have been taken to develop inhibitors of this Hospital Clinic Animal Experimentation Committee, according signaling pathway (6). Akt acts as a central node of this pathway, to Spanish official regulations. and hyperactivated Akt is a common feature observed in human solid tumors (3, 5). Therefore, Akt has been proposed as an Pharmacokinetics of ABTL0812 in mice and rats interesting target in cancer therapy. Two classes of Akt inhibitors ABTL0812 was orally (gavage, 100 mg/kg) and intravenously are now in clinical development: ATP-competitive inhibitors (10 mg/kg) administered to male CD-1 mice. ABTL0812 was (such as GSK690693) which target active Akt, and allosteric quantified in plasma by validated LC/MS-MS methods and phar- inhibitors (such as MK-2206 and perifosine, currently in phase macokinetic parameters were calculated with WinNonlin II and phase III clinical trials, respectively) that by binding the PH software. domain of Akt prevent its activation by upstream kinases (3, 7). Here we present ABTL0812, a first-in-class molecule with anti- Clinical trial and isolation of PBMC from patients tumor activity which is currently in phase I/Ib clinical trials in ABTL0812 is currently in clinical evaluation in a phase I/Ib trial patients with advanced solid tumors. We provide evidences show- in patients with advanced solid tumors. This study was approved ing that ABTL0812 inhibits Akt phosphorylation by upregulating by the local Ethics Committee of the Hospital Clinic Barcelona the expression of TRIB3,a pseudokinase that binds and inhibits Akt, and the Spanish Agency of Medicines. The trial is registered at leading tomTORC1 inhibition and autophagy-mediated cell death. www.clinicaltrials.gov (NCT02201823). ABTL0812 was administered daily by the oral route at a dose of 1,000 mg twice daily for Methods a period of 28 days. Blood samples for biomarker study were obtained by venipuncture before and after ABTL0812 treatment. Cell culture, viability assay, transfection, and lysis PBMC were isolated by Ficoll (GE-Healthcare) density centrifu- Human cancer cell lines were purchased from ATCC (2008; þ þ gation, lysed in RIPA buffer, sonicated, and stored at 20 C. ATCC-authentication by isoenzymes analysis). Atg5 / and Atg5 / T-large antigen–transformed MEFs were donated by Dr. þ þ Mizushima (Tokyo Medical University, Tokyo, Japan). TRB3 / Statistical analysis in vitro and TRB3 / RasV12/E1A–transformed Mouse embryonic fibro- All data were assessed using one-way ANOVA followed blasts (MEF) have been described before (8). Cell lines were by Bonferroni multiple comparison test. Tumor volumes of mice t cultured as recommended and transfected using Lipofectamine- were compared using the ANOVA followed by test. Statistical fi 2000. Cell viability was determined by the MTT (3-(4, 5-dimethyl- signi cance between the groups was assessed with the log-rank fi 2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium Blue) reduction assay test (GraphPad). Levels of statistical signi cance were set at P (9). Cells were lysed in ice-cold RIPA buffer supplemented with 1 0.05. mmol/L sodium-orthovanadate, 50 mmol/L NaF, and 5 mmol/L sodium-pyrophosphate, sonicated and stored at 20C. Results ABTL0812 has anticancer activity in vitro and in vivo Immunoblotting and immunoprecipitation A novel class of chemically modified lipid-derived small mole- Immunoblot analyses were performed following standard cules was selected on the basis of two phenotypic assays: anti- procedures as described (10) and antibodies listed in the Sup- proliferative effect on tumor cells and low toxicity after high doses

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Figure 1. ABTL0812 induces cancer cell death in vitro and reduces tumor growth in human lung and pancreatic xenografts. A, chemical structure of ABTL0812. B, ABTL0812 cell viability assays in human cancer cell lines. IC50 values were obtained in MTT assays after exposure to ABTL0812 for 48 hours. Values are the mean SD of three separate determinations. (Continued on the following page.)

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administration in rodents. From this class, ABTL0812 was selected also induced hallmarks of autophagy in vivo, as tumors from A549 for further preclinical development. The formula for ABTL0812 is xenograft mice treated with ABTL0812 showed an increase in LC3- 2-hydroxylinoleic acid (Fig. 1A). II levels, compared with mice treated with vehicle (Fig. 2C). ABTL0812 reduced viability of different human cancer cell lines Next, we investigated the role of autophagy in the cell death tested with IC50 values of 20 to 60 mmol/L (Fig. 1B). Real-time cell induced by ABTL0812. Pharmacologic inhibition of autophagy analyses further demonstrated the antiproliferative effect of was achieved by treating cells with a combination of the lyso- ABTL0812 (Supplementary Fig. S1). ABTL0812 also induced cell somal protease inhibitors E64d and Pepstatin-A (PA) that block death, which was confirmed by propidium iodide staining (Sup- the final step of autolysosomal degradation. Treatment of cells plementary Fig. S2). Importantly, ABTL0812 induced death in with E64dþPA prevented ABTL0812-induced cell death and cancer cells but not in normal cells. Compared with LN-18 resulted in an enhancement of LC3-II accumulation in cells glioblastoma cells, primary astrocytes were resistant to ABTL0812 treated with ABTL0812 (Fig. 2D), indicating that ABTL0812 treatment at the highest ABTL0812 concentration tested (200 induces dynamic autophagy in cancer cells. Finally, we evaluated mmol/L; Fig. 1C). the activity of ABTL0812 in oncogene-transformed embryonic To address ABTL0812 pharmacokinetics, we administered the fibroblasts derived from Atg5 / (autophagy-deficient) mice. compound to mice by oral and intravenous routes. ABTL0812 Atg5 is an essential protein for autophagosome formation showed an excellent bioavailability by oral route (F:103%), (14). Transformed Atg5 / MEF cells were more resistant to þ/þ with rapid oral absorption (tmax:30 minutes), high pick concen- ABTL0812-induced cell death than transformed Atg5 MEF trations (Cmax:2.0 mg/mL), and wide volume of distribution cells, and did not activate autophagy in response to ABTL0812 (Vss:10.4 L/kg). Clearance was also high (Cl: 82 mL/minute/kg). treatment (Fig. 2E). Taken together, these results show that Overall, the compound was well tolerated and animals appeared autophagy mediates ABTL0812-induced cancer cell death. healthy with no clinical signs of distress, local or systemic toxicity. fi in To test the ef cacy of ABTL0812 in inhibiting tumor growth ABTL0812 inhibits the Akt/mTOR axis vivo , we used human lung and pancreatic xenograft models. A549 Autophagy induction is often consequential to the inhibition human lung cancer xenografts of nude mice were treated with of mTOR signaling, as mTORC1 acts as a central regulator in ABTL0812 or the standard-of-care docetaxel. ABTL0812 inhibited autophagy induction (15). We therefore investigated whether fi tumor progression with an ef cacy similar to docetaxel (Fig. 1D). ABTL0812-induced autophagy and cancer cell death occurred via Moreover, ABTL0812 lacked the toxic effects observed for doc- mTORC1 inhibition, by measuring levels of activation of com- etaxel, as shown by body weight measurements. Similarly, ponents of the Akt/mTORC1 signaling pathway. A549 and Mia- ABTL0812 also inhibited the growth of MiaPaCa-2 human pan- PaCa-2 cells showed measurable phosphorylation levels for all creatic cancer xenografts in nude mice, without affecting body the proteins of Akt/mTORC1 axis in basal conditions. ABTL0812 weight (Fig. 1E). treatment reduced phosphorylation of ribosomal-S6 kinase and of ribosomal-S6 protein (a well-established S6K substrate) in a ABTL0812 induces autophagy-mediated cell death concentration-dependent manner (Fig. 3). Next, we investigated the type of cell death induced by Protein kinase Akt modulates the activation of mTORC1 by ABTL0812 in A549 and MiaPaCa-2 cells. ABTL0812 did not phosphorylating and inactivating the TSC2 and PRAS40 proteins induce nuclear fragmentation/condensation, or caspase-3 activa- that act as repressors of mTORC1 activity (1). Thus, Akt inhibition tion (Supplementary Fig. S3). Treatment with staurosporine results in impaired mTORC1 activity and activation of autophagy. resulted in typical features of apoptosis, showing that A549 and ABTL0812 induced Akt inhibition in A549 and MiaPaCa-2 cells in MiaPaCa-2 cells are apoptosis competent. These results indicate a dose-dependent manner, by reducing phosphorylation of the that ABTL0812 fails to induce apoptosis. In turn, several evidences two critical residues involved in Akt activation, Thr308 and indicated that ABTL0812-induced cell death is mediated by Ser473. Consequently, ABTL0812 also reduced phosphorylation autophagy. of the Akt substrates PRAS40 and TSC2 (Fig. 3). These results show Two of the hallmarks of autophagy are the conversion of the that ABTL0812 induced inhibition of the Akt/mTORC1 axis in soluble form of LC3 to a lipidated form associated to autophago- cancer cells. somes (LC3-II), and the elimination of autophagosome cargo proteins such as p62 (13). In A549 and MiaPaCa-2 cells, ABTL0812 binds and induces PPARa and PPARg ABTL0812 induced the appearance of LC3-II and a decrease on transcriptional activities, which mediate in ABTL0812-induced p62 protein in a concentration-dependent manner (Fig. 2A). We cancer cell death also observed the occurrence of LC3-positive dots in cells treated To identify the molecular targets of ABTL0812, we performed in with ABTL0812, which indicates association to autophagosomes silico high-throughput screening comparing ABTL0812 chemical (Supplementary Fig. S4). Electron microscopy analysis of structure against the ChEMBL15 database (https://www.ebi.ac. ABTL0812-treated A549 and MiaPaCa-2 cells revealed the pres- uk/chembldb/), following the similar property and active analog ence of double membrane vacuolar structures with the morpho- principles (16). This approach allowed the identification of logic features of autophagosomes (Fig. 2B). Moreover, ABTL0812 several ABTL0812 target candidates, including PPARa and PPARg.

(Continued.) C, LN-18 glioblastoma cells or primary rat astrocytes were incubated with ABTL0812, and 48 hours later cell viability was monitored by MTT assays. Similar results were obtained in three separate determinations. D and E, ABTL0812 inhibits tumor growth in lung (D) and pancreatic (E) cancer xenograft models. Athymic nude mice injected with A549 or MiaPaCa-2 cells were treated with vehicle or ABTL0812 by oral gavage. Docetaxel was administered by intraperitoneal injection. Tumor growth and weight variation curves are shown. Results are the mean SD of ﬁve mice in each group. Because of toxicity in 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.

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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/L ABTL0812 for 12 hours, lysed and levels of the autophagy-marker proteins LC3 and p62 visualized by immunoblotting. B, representative electron microscopy microphotographs showing autophagosomes (right) in cells treated with 50 mmol/L ABTL0812 for 10 hours. C, total protein extracted from A549 xenograft tumors (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 were preincubated with vehicle or a combination of lysosomal protease inhibitors E64d (10 mmol/L) and Pepstatin-A (PA, 10 mg/mL), before treatment with 50 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 from þ þ ABTL0812 treated cells. Cells were lysed and LC3 lipidation was visualized by immunoblotting. E, Atg5 / and Atg5 / MEFs were treated with vehicle or ABTL0812 þ þ and cell viability determined 24 hours later. Values are the mean SD of three different experiments. , P < 0.05; , P < 0.001 from Atg5 / MEFs.

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Finally, we performed cell viability assays in the presence of PPARa/g antagonists. In A549 and MiaPaCa-2 cells, preincuba- tion with PPARa antagonist GW6471 or PPARg antagonist GW9662 prevented cell death induced by ABTL0812 (Fig. 4C), indicating that PPAR receptors mediate in ABTL0812-induced cell death.

ABTL0812 activates PPARa- and PPARg-dependent expression of TRIB3 Koo and colleagues showed that PPARs regulate the expression of 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 an inactive form of Akt (8, 19). Thus, we hypothesized that ABTL0812 could inhibit Akt (and downstream mTORC1) by upregulating TRIB3 via PPARa/g activation. In A549 and Mia- PaCa-2 cells, ABTL0812 induced a quick and robust increase in TRIB3 protein levels after 12 hours of treatment (Fig. 5A), that correlated with an increase in gene transcription (Fig. 5B) and qPCR-measured TRIB3 mRNA levels (Fig. 5C). Although we Figure 3. cannot discard an effect of ABTL0812 on TRIB3 stability, these ABTL0812 inhibits the Akt/mTORC1 axis. A549 and MiaPaCa-2 cells treated results indicate that ABTL0812 upregulates TRIB3 gene transcrip- with vehicle (0) or ABTL0812 (mmol/L) for 24 hours were lysed, and levels of tion. Furthermore, treatment with PPARa and/or PPARg inhibi- phosphorylated proteins were analyzed by immunoblotting. Levels of total tors GW6471 and GW9662, respectively, completely abolished proteins and actin were used as control. Similar results were obtained in three the ABTL0812-induced upregulation of TRIB3 gene expression separate experiments. (Fig. 5C). Interestingly, treatment with either PPARa or PPARg agonists resulted in increased TRIB3 expression and reduced Akt phosphorylation (Fig. 5D). However, it was necessary to combine Of note, a possible interaction with PPARb/d was not found. treatment with both agonists to obtain similar levels of inhibition Putative ABTL0812-binding molecules identified in this screening of Akt (phospho-PRAS40) and mTORC1 activities (phospho-S6K were further investigated through docking analysis. Figure 4A and phospho-S6) than those induced by ABTL0812. Altogether, shows the molecular model of ABTL0812 into the ligand-binding our results demonstrate that ABTL0812 induces TRIB3 gene pocket of PPARg. In this model, ABTL0812 may bind the PPARg expression via activation of PPARa and PPARg activities. ligand-binding domain (LBD) through its carboxylate group, which forms hydrogen bonds with the side of key amino acid residues (Ser289, His323, Tyr473, and His449), as it has been ABTL0812 inhibits Akt and mTORC1 via TRIB3 shown for other PPARg agonists such as rosiglitazone (17). The To investigate the role of upregulated TRIB3 on Akt activation, 2-hydroxyl group of ABTL0812 may also form hydrogen bonds we overexpressed increasing amounts of TRIB3 in A549 and with residues Cys285 and Ser289, which are also conserved in the MiaPaCa-2 cells. We observed reduced phosphorylation of Akt PPARa LBD. This molecular model proposes that ABTL0812 and Akt substrate PRAS40 that inversely correlated with TRIB3 – might establish interactions similar to the specific PPARg agonist expression (Fig. 6A). This was probably due to a TRIB3 Akt rosiglitazone, thus predicting that ABTL0812 could act as PPAR physical interaction, because ABTL0812 treatment increased the agonist. Indeed, in vitro radioligand displacement assays using amount of Akt coimmunoprecipitated with TRIB3 (Fig. 6B). fi purified proteins showed that ABTL0812 binds PPARa and PPARg Moreover, unlike wild-type transformed MEF, TRIB3-de cient MEF cells did not show Akt (Ser473) or mTORC1 (pS6) inhibi- ligand-binding pockets with Ki values of 7.1 mmol/L and 4.7 mmol/L, respectively (Supplementary Fig. S5). tion, or induction of autophagy (LC3-II marker) in response to Next, we investigated the functional relevance of this interac- ABTL0812 treatment (Fig. 6C). Cell viability assays showed that fi tion, performing luciferase-based gene reporter assays in cells TRIB3-de cient MEFs are resistant to ABTL0812-induced cell transfected with a PPAR-response element (PPRE) fused to lucif- death, unlike wild-type transformed MEFs (Fig. 6D). These results erase construct. In A549 and MiaPaCa-2, ABTL0812 induced support a pivotal role of TRIB3 on the impairment of the Akt/ approximately 5-fold PPAR transcriptional activity, similarly to mTORC1 axis and on the autophagy-mediated cancer cell death the specific agonists WY-14643 (PPARa) or rosiglitazone (PPARg; observed in response to ABTL0812 treatment. Supplementary Fig. S6). To determine whether ABTL0812- induced PPAR activation was mediated by the PPAR isoforms ABTL0812 induces TRIB3 upregulation and Akt inhibition a and/or g, we performed analogous gene reporter assays in cells in vivo transiently transfected with plasmids encoding each isoform. To determine the in vivo relevance of our findings, we next ABTL0812-induced PPAR transcriptional activity was higher in investigated the effect of ABTL0812 in A549 human lung and cells overexpressing PPARa or PPARg proteins, compared with MiaPaCa-2 human pancreatic cancer xenografted mice (Fig. 1C those having the endogenous proteins only (Fig. 4B), suggesting and D). Immunohistochemical and immunoblotting analyses that ABTL0812 functionally interacts with both receptors in cells. showed that ABTL0812-induced upregulation of TRIB3 protein

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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 plasmids were cotransfected with plasmids encoding empty backbone or PPARa or PPARg. Sixteen hours posttransfection, cells were treated with vehicle or 50 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 with vehicle (white columns), 0.2 mmol/L GW6471 (PPARa-antagonist) or 1 mmol/L GW9662 (PPARg-antagonist) during 2 hours, and then treated with 50 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. and inhibition of Akt phosphorylation (Ser473) in A549 and treatment with ABTL0812 resulted in a marked reduction on Akt MiaPaCa-2 tumors (Fig. 6E). These data, together with that pre- phosphorylation in PBMCs from 3 patients (Fig. 6F). We could sented in Fig. 2 showing that ABTL0812 induces autophagy in not detect TRIB3 protein in PBMCs, using commercially available A549 xenografts, suggest that the antitumor activity of ABTL0812 anti-TRIB3 antibodies. However, parallel qPCR analysis showed a in vivo relies on inhibition of Akt and induction of autophagy- signiﬁcant increase in TRIB3 mRNA levels in human PBMCs mediated cell death. treated in vitro with ABTL0812 (Supplementary Fig. S7), thus suggesting that ABTL0812 also induces Akt inhibition in humans Akt phosphorylation is a pharmacodynamic marker in patients through upregulation of TRIB3. treated with ABTL0812 Finally, we evaluated the activity of ABTL0812 in some patients Discussion included in phase I/Ib clinical trials (NCT02201823), monitoring Akt phosphorylation levels in PBMCs. We collected blood sam- Here, we present the mechanism of action of ABTL0812, a novel ples, pretreatment and at different days of chronic treatment with and ﬁrst-in-class antitumor drug which is currently in phase I/Ib 1,000 mg of ABTL0812, a dose that was safe and well tolerated clinical trials in patients with advanced solid tumors. ABTL0812 is (preliminary data not shown). Our preliminary results show that a polyunsaturated fatty acid derivative, small molecule that

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Figure 5. ABTL0812 induces upregulation of the Akt endogenous inhibitor TRIB3 through PPARa/g activation. A, cells treated with ABTL0812 for the indicated times were lysed, and levels of TRIB3 and actin analyzed by immunoblotting. B, cells were transfected with luciferase-TRIB3 promoter reporter and pRL-CMV-Renilla plasmids. Sixteen hours posttransfection, cells were treated with vehicle or 100 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. 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 treated for 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 agonist rosiglitazone (1 mmol/L) for 24 hours, lysed and proteins analyzed by immunoblotting.

induces cell death in a broad panel of cancer cell lines, whereas it type, stage, and genetic context, as well as on the duration and does not affect cell viability of nontumorogenic cells. ABTL0812 strength of the triggering stimuli (15, 20). Our results demonstrate also inhibits tumor growth in human lung and pancreatic tumor that ABTL0812 induces autophagy-mediated cancer cell death in xenografts. Herein, we elucidated a novel cell signaling pathway vitro and in vivo, without activating cellular apoptosis. These through which ABTL0812 induces autophagic cell death. We results are in agreement with those reporting that polyunsaturated showed that ABTL0812 induces upregulation of the TRIB3 pseu- fatty acid and derivatives exert their antiproliferative action dokinase through activation of PPARa and PPARg transcription through activation of autophagy (21–23). factors. Upregulated TRIB3 binds to Akt and prevents its activation The majority of current anticancer treatments activate apopto- by PDK1 and mTORC2 upstream kinases, resulting in inhibition sis, and resistance to chemotherapy is a major challenge in cancer of the Akt/mTORC1 axis and, therefore, autophagy-mediated cell (24). Autophagy-mediated cell death has emerged as an alterna- death (Fig. 6G). tive to kill cancer cells without inducing resistance to apoptosis Autophagy is an essential process that consists of selective inducer drugs (25). mTORC1 plays a central role in regulating degradation of cellular components, assuring the maintenance cellular autophagy, and mTORC1 inhibitors induce autophagy- of cellular homeostasis via lysosomal degradation pathways (13). mediated cell death in many systems, such as AZD8055 in The role of autophagy in cancer is still far from clear, as it can act as hepatocellular carcinoma (26) or Ku0063794 and temsirolimus a tumor suppressor and tumor promoter, depending on tumor in renal carcinoma (27). On the other hand, mTORC1 activation

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Inhibition of Akt/mTORC1 Axis by TRIB3 Pseudokinase

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 encoding human-TRIB3 were lysed and levels of proteins monitored by immunoblotting. B, immunoprecipitation of Akt from MiaPaCa-2 lysates after treatment with vehicle 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 ABTL0812 for 24 hours, and levels of the indicated proteins were analyzed by immunoblotting. D, wild-type and TRIB3-KO MEFs were treated with ABTL0812 for 24 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 and MiaPaCa-2 tumors were collected after sacrifice, and TRIB3 was evaluated by immunohistochemical analysis (left; scale bars: 100 mm). Levels of phospho-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 after 1,000 mg bid ABTL0812 oral chronic treatment. Protein extracts were analyzed by immunoblotting for phospho-Akt-Ser473 and total Akt. Upper graphs show the quantification of levels of phospho-Akt normalized with Akt protein, estimated by densitometric analysis (day 1 ¼ 100%). G, a model showing the mechanism of action of ABTL0812. www.aacrjournals.org Clin Cancer Res; 2016 OF9

Erazo et al.

is frequently associated with resistance to antitumor drugs (6). As shown that TRIB3 can interact with Akt, preventing its phosphor- ABTL0812 is a potent inhibitor of the Akt/mTORC1 axis, its ylation by upstream kinases and resulting in suppression of the administration in combination with standard chemotherapeutic insulin pathway (16, 19, 42). Here we show that ABTL0812- drugs might prove effective in therapy-resistant or apoptosis- evoked TRIB3 binds Akt, and that TRIB3 overexpression in cells refractory tumor. This has been shown for the mTORC1 inhibitor resulted in Akt inhibition. Moreover, we also found that treatment everolimus (RAD001), which sensitizes papillary thyroid cancer with ABTL0812 enhances the interaction between TRIB3 and Akt, cells to treatment with doxorubicin by an autophagy-mediated in line with previous data obtained in glioma cells and oncogene- mechanism (28). transformed MEFs, where a similar increase in TRIB3 levels and Here we present in vitro and in vivo evidences showing that TRIB3-Akt interaction occur upon exposure to D9-tetrahydrocan- ABTL0812 binds to and activates PPARa and PPARg transcriptional nabinol (THC; refs. 12). TRIB3 interacts more strongly with activities, and that pharmacologic inhibition of both receptors inactive/dephosphorylated Akt than active Akt (M. S., unpub- prevents ABTL0812-induced cancer cell death, suggesting that lished results), therefore it is likely that overexpressed TRIB3 PPARa/g are the primary molecular targets of ABTL0812 in the could trap the inactive form of Akt, preventing its activation/ cell. PPARs are nuclear receptors that heterodimerize with the phosphorylation by upstream kinases. Whether TRIB3 masks the retinoid-X receptors to modulate gene transcription. Mammalian phosphorylatable residues in Akt (Thr308 and Ser473), induces a cell expresses three PPAR isoforms, PPARa,PPARb/d, and PPARg conformational change that impairs recognition by upstream (29). Using an in silico analysis, we identified PPARa and PPARg as kinases PDK1 and mTORC2, or hampers the binding of the Akt targets of ABTL0812, but not PPARb/d. Several authors have shown PH domain to PIP3 remains to be clarified. On the other hand, it is that polyunsaturated and derivative long fatty acids activate PPARs also likely that, in addition to changes in TRIB3 protein levels, transcriptional activities (21, 30). Interestingly, the docking experi- there could exist additional mechanisms involved in regulating ments performed in our study show that the a-hydroxyl group in the interaction of TRIB3 with its protein targets, and specifically ABTL0812 forms two extra hydrogen bonds with the critical resi- with Akt. Posttranslational modifications of TRIB3, yet to be dues Cys285/Ser289 in PPARg LBD (also conserved in the PPARa described, and/or changes in the interaction with other members LBD), compared with the nonhydroxylated linoleic acid. Accord- of the Tribbles family that participate in modulating these inter- ingly, ABTL0812 showed higher PPARg agonistic activity (Ki 5 actions, are two plausible possibilities that are currently investi- mmol/L) than linoleate (79 mmol/L) or the PPARg natural agonists gated in our laboratories. flavonoids such as kaempferol (30 mmol/L; ref. 30). Our results indicate that TRIB3 plays a central role in the PPARs regulate the expression of many genes involved in mechanism of action of ABTL0812, as it has been proposed for glucose and lipid metabolism (29, 31). Interestingly, activation other antitumoral drugs, such as THC in glioma and hepatocel- of PPARg reduces cell proliferation and invasion, and enhances lular carcinoma tumors (43, 44) or salinomycin in lung cancer apoptosis in different cancer models such as breast and hepato- cells (45). Interestingly, these drugs also induce autophagy, as it carcinoma (21, 32), whereas activation of PPARa inhibits does ABTL0812, suggesting that TRIB3 upregulation effectively tumor growth and angiogenesis in mouse models (33–35). provokes a robust inhibition of the Akt/mTORC1 axis in vivo. THC Therefore, activators of PPARa or PPARg have been proposed and salinomycin exert their action by stimulation of endoplasmic as anticancer drugs, and specific synthetic PPARa or PPARg reticulum stress and then activating ATF4/CHOP transcription agonists are in clinical trials for treating cancer, such as the PPARg factors that regulate TRIB3 gene. In our study, we identify a new agonist efatutazone or the PPARa agonist fenofibrate, in patients route that links PPARa/g activation, inhibition of Akt/mTORC1 with liposarcoma (NTC02249949) or multiple myeloma via TRIB3 and cancer cell death. (NCT01965834), respectively. ABTL0812, by activating PPARa There are contradictory reports in the literature regarding the role and PPARg, might well synergize the anticancer activity of each of TRIB3 in human cancer. Some laboratories reported elevated receptor. Importantly, single treatment of the cancer cells used in TRIB3 mRNA levels which correlated with bad prognosis in colo- our study with either PPARa or PPARg agonist induced partial rectal cancer (46) and breast cancer patients (47). On the other inhibition of mTORC1 activity, whereas combined treatment hand, high TRIB3 protein levels have been associated with good with both agonists resulted in a robust mTORC1 inhibition, prognosis sensitivity to radiotherapy in breast cancer patients (48). similar to that obtained for ABTL0812 treatment (Fig. 5D). Given As TRIB3 is a very stable protein (49), the discrepancies observed the fact that a new generation of dual PPARa/g agonists has shown between TRIB3 mRNA and protein levels in human breast cancer promising anticancer activity (i.e., TZD18 in breast cancer; ref. 36), prognosis might be due to posttranslational modifications, still to it will be of interest to investigate whether PPARa/g mechanism of be described, that might modulate the ability of TRIB3 to interact action also involves inhibition of the Akt/mTORC1 axis. with other proteins that regulate tumor growth. In this study, Our most striking result is the observation that upregulation of however, we show that ABTL0812 treatment induces a robust Tribbles pseudokinase-3, through activation of PPARa/g recep- increase in TRIB3 transcription and mRNA and protein levels, which tors, plays a crucial role in the anticancer activity of ABTL0812. correlate with cancer cell death and tumor growth inhibition. Our TRIB3 is a highly conserved protein which lacks critical catalytic results are in agreement with a recent report showing that genetic residues for binding ATP and therefore lacks kinase activity (37). inhibition of TRIB3 enhances tumorogenesis in different cancer Together with TRIB1 and TRIB2 proteins, TRIB3 define the Trib- models (8), and support a role for TRIB3 as a tumor suppressor. bles subfamily of pseudokinases, which was first described in Hyperactivation of the Akt/mTORC1 axis is observed in the Drosophila as regulators of cell proliferation and migration during majority of human cancers and blocking this pathway is an development (38). As it happens for other pseudokinases, TRIB3 important anticancer strategy (5). We have shown here the activity exerts its function in the cell interacting with several proteins, such of ABTL0812 in a panel of cancer cell lines that express high levels as the transcription factors SMAD3 (39) and ATF-4 (40), or of phosphorylated/active Akt. Importantly, we have preliminary members of the MAPK family (41). Of note, few authors have observed activity of ABTL0812 in patients. ABTL0812 treatment

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Inhibition of Akt/mTORC1 Axis by TRIB3 Pseudokinase

impaired Akt phosphorylation in PBMCs from patients included Writing, review, and/or revision of the manuscript: M. Lorente, in the ongoing phase I clinical trial (Fig. 6F). Therefore, Akt A. Lopez-Plana, P. Fernandez-Nogueira, G. Fuster, J. Espadaler, J. Hernandez- phosphorylation will be used as a surrogated pharmacodynamic Losa, J.R. Bayascas, M. Cortal, L. Vidal, P. Gascon, M. Gomez-Ferreria, J. Alfon, G. Velasco, C. Domenech, J.M. Lizcano biomarker, for estimating the recommended dose for the forth- Administrative, technical, or material support (i.e., reporting or organizing coming phase II trials. We have preliminarily observed increased data, constructing databases): J.M. Lizcano TRIB3 mRNA levels in human PBMCs treated with ABTL0812 Study supervision: P. Gascon, G. Velasco, J.M. Lizcano (Supplementary Fig. S7), suggesting that TRIB3 qPCR analysis could be also used to monitor the biologic activity of ABTL0812 in Acknowledgments humans. Finally, and given its unique mechanism of action, it will The authors thank Pablo Escriba and Xavier Busquets (Universitat Illes be important to evaluate ABTL0812 in combination with stan- Balears, Spain) for the preliminary identification of ABTL0812 as an anticancer dard chemotherapeutical compounds as well as with other target agent (patent number WO/2010/106211) and Giovani Cincilla, Emiliana D'Oria, and Oscar Villacanas~ (Intelligent Pharma SL) for in silico analysis, Luis therapies. We foresee ABTL0812-based therapies will contribute Botella and Javier Soto (Medalchemy) for the synthesis of ABTL0812, and to improve patients' treatment and quality of life. Washington Biotechnology Inc. for preliminary preclinical studies. The authors fl also thank Victor Yuste for helpful discussions, Cristina Gutierrez for tissue Disclosure of Potential Con icts of Interest culture assistance, and Servei de Genomica i Informatica from the UAB. J. Espadaler has ownership interest (including patents) in and is a consultant/ advisory board member for Ability Pharmaceuticals SL. J.M. Lizcano is a consultant/advisory board member for Ability Pharmaceuticals. No potential Grant Support conflicts of interest were disclosed by the other authors. This work was supported by grants from the Government of Catalonia (ACCIO/FINEBT10-1-0047), and from the Spanish Ministry of Economy Authors' Contributions and Competitiveness (MINECO), (CDTI/PID/IDI-20101630, Genoma ~ Conception and design: M. Salazar, J. Espadaler, M. Gomez-Ferreria, J. Alfon, Espana/INNOCASH/2011196, ENISA/EBT2012/100375, INNPACTO/IPT- C. Domenech, J.M. Lizcano 2012-0614-010000). J. Alfon and M. Gomez-Ferreria were funded by Torres- Development of methodology: T. Erazo, M. Lorente, A. Lopez-Plana, Quevedo grant (MINECO), and M. Cortal by an Inncorpora grant (MINECO). P. Munoz-Guardiola,~ P. Fernandez-Nogueira, G. Fuster, J. Hernandez-Losa, Work in G. Velasco's laboratory was supported by grants from Spanish Ministry J.M. Lizcano of Economy and Competitiveness (MINECO), Fondo Europeo de desarrollo Acquisition of data (provided animals, acquired and managed patients, Regional (FEDER) (PI12/02248 and PI15/00339) and Fundacion Mutua ~ provided facilities, etc.): T. Erazo, M. Lorente, P. Munoz-Guardiola,~ Madrilena (AP101042012). P. Fernandez-Nogueira, J.A. García-Martínez, P. Bragado, M. Salazar, The costs of publication of this article were defrayed in part by the payment of advertisement J. Hernandez-Losa, J.R. Bayascas, L. Vidal, G. Velasco page charges. This article must therefore be hereby marked in Analysis and interpretation of data (e.g., statistical analysis, biostatistics, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. computational analysis): T. Erazo, M. Lorente, A. Lopez-Plana, P. Fernandez-Nogueira, J.A. García-Martínez, P. Bragado, M. Salazar, Received July 30, 2015; revised November 20, 2015; accepted November 30, J. Espadaler, M. Cortal, J. Alfon,C.Dom enech, J.M. Lizcano 2015; published OnlineFirst December 15, 2015.

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The New Antitumor Drug ABTL0812 Inhibits the Akt/mTORC1 Axis by Upregulating Tribbles-3 Pseudokinase

Tatiana Erazo, Mar Lorente, Anna López-Plana, et al.

Clin Cancer Res Published OnlineFirst December 15, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-15-1808

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