París-Coderch et al. Cell Death and Disease (2020)11:773 https://doi.org/10.1038/s41419-020-02986-w Cell Death & Disease ARTICLE Open Access The antitumour drug ABTL0812 impairs neuroblastoma growth through endoplasmic reticulum stress-mediated autophagy and apoptosis Laia París-Coderch 1, Aroa Soriano 1, Carlos Jiménez 1, Tatiana Erazo2, Pau Muñoz-Guardiola2,3, Marc Masanas1, Roberta Antonelli1, Ariadna Boloix 1, José Alfón3, Héctor Pérez-Montoyo3, Marc Yeste-Velasco3, Carles Domènech3, Josep Roma 1, Josep Sánchez de Toledo1,4, Lucas Moreno1,4, José M. Lizcano 2, Soledad Gallego 1,4 and Miguel F. Segura 1 Abstract Neuroblastoma is the leading cause of cancer death in children aged 1 to 4 years. Particularly, five-year overall survival for high-risk neuroblastoma is below 50% with no curative options when refractory or relapsed. Most of current therapies target cell division and proliferation, thereby inducing DNA damage and programmed cell death. However, aggressive tumours often present alterations of these processes and are resistant to therapy. Therefore, exploring alternative pathways to induce tumour cell death will provide new therapeutic opportunities for these patients. In this 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; study we aimed at testing the therapeutic potential of ABTL0812, a novel anticancer drug that induces cytotoxic autophagy to eliminate cancer cells, which is currently in phase II clinical trials of adult tumours. Here, we show that ABTL0812 impaired the viability of clinical representative neuroblastoma cell lines regardless of genetic alterations associated to bad prognosis and resistance to therapy. Oral administration of ABTL0812 to mice bearing neuroblastoma xenografts impaired tumour growth. Furthermore, our findings revealed that, in neuroblastoma, ABTL0812 induced cancer cell death via induction of endoplasmic reticulum stress, activation of the unfolded protein response, autophagy and apoptosis. Remarkably, ABTL0812 potentiated the antitumour activity of chemotherapies and differentiating agents such as irinotecan and 13-cis-retinoic acid. In conclusion, ABTL0812 distinctive mechanism of action makes it standout to be used alone or in combination in high-risk neuroblastoma patients. Introduction countries and ~15% of all cancer-related deaths in chil- Neuroblastoma is an embryonic neuroendocrine dren. In Europe, the estimated annual incidence is ~1000 tumour of the sympathetic nervous system, representing new cases per year, of which 60% are classified as high- 8–10% of all diagnosed childhood cancers in western risk, with a 5-year overall survival below 50%1–3. Fur- thermore, a significant proportion of patients will suffer major treatment-associated long-term side effects and Correspondence: Miguel F. Segura (miguel.segura@vhir.org) 1 life-long disabilities2. Translational Research in Child and Adolescent Cancer, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain Autophagy is an evolutionary-conserved catabolic pro- 2 Protein Kinases and Signal Transduction Laboratory, Institut de Neurociències cess that recycles unnecessary or dysfunctional cellular and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma components4. In cancer, autophagy has been proposed to de Barcelona, Bellaterra, Spain Full list of author information is available at the end of the article suppress malignant transformation by controlling cell Edited by G.M. Fimia © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association París-Coderch et al. Cell Death and Disease (2020)11:773 Page 2 of 10 growth, degradation of oncogenic/damaged proteins, inactivated foetal bovine serum (FBS, South America preservation of genetic/genomic stability and induction of Premium, Bio-west), 1% of insulin–transferrin–selenium autophagic cell death among other mechanisms. How- Supplement (Thermo Fisher Scientific), 100 U/mL peni- ever, in established tumours, autophagy may also promote cillin, 100 μg/mL streptomycin (Thermo Fisher Scientific) tumour progression and resistance to therapy through and 5 μg/mL plasmocin (InvivoGen, San Diego, CA, conferring resistance to hypoxia and nutrient deprivation, USA). All cultures were maintained at 37 °C in a saturated induction of dormancy; disabling immunosurveillance or atmosphere of 5% CO2. All cell lines were periodically facilitating EMT transition and dissemination [reviewed tested for mycoplasma contamination. in refs. 5,6]. In neuroblastoma, several compounds have already shown to exert antitumour activity through the Reagents activation of autophagy7–10. ABTL0812 was provided by Ability Pharmaceuticals The novel compound ABTL0812 is the sodium salt (Cerdanyola del Vallès, Spain) and cis-Diammineplatinum formulation derivative from 2-hydroxy-linoleic acid, an (II) dichlorideDNA (Cisplatin) was purchased from essential polyunsaturated ω6 fatty acid made up of eigh- Sigma-Aldrich (St. Louis, MO, USA). Therapeutic agents teen carbons (chemical name: sodium 2-hydro- doxorubicin hydrochloride, irinotecan (SN-38), topote- xylinoleate). ABTL0812 triggers cytotoxic autophagy can, cyclophosphamide monohydrate and 13-cis-retinoic through inhibition of the AKT/mTOR pathway11 and acid (Isotretinoin) were purchased from Selleckchem through the sustained induction of endoplasmic reticu- (Houston, TX, USA). Autophagy inhibitors E64d lum (ER) stress and activation of the unfolded protein (Cysteine-catepsines inhibitor), Pepstatin A (Aspartic- response (UPR)12. Of note, these two pathways have been proteases inhibitor) and the pan-caspase inhibitor QVD- shown to modulate the expression or stability of MYCN, a OPh were purchased from Sigma-Aldrich. key undruggable genetic driver in neuroblastoma13,14. Therefore, we decided to test the therapeutic potential of Cell proliferation assays ABTL0812 in neuroblastoma. Neuroblastoma cells were seeded in 96-well plates at ABTL0812 successfully completed a Phase I/Ib trial in 6–16 × 103 cells/well (n = 6/condition) and 24 h later patients with advanced solid tumours (NCT02201823), treated with the designated drugs diluted in culture showing a good safety and tolerability profile. Moreover, medium supplemented with 0.5% FBS. At the indicated first signs of efficacy were observed15. These satisfactory times, cells were fixed with 1% glutaraldehyde (Sigma- results led to the ongoing Phase I/II trials in patients with Aldrich) and stained with 0.5% crystal violet (Sigma- advanced endometrial cancer and squamous non-small Aldrich). Crystals were dissolved in 15% acetic acid cell lung cancer, where ABTL0812 is given at first line in (Thermo Fisher Scientific) and absorbance was measured combination with chemotherapy (NCT03366480). at 590 nm using an Epoch Microplate Spectrophotometer Here, we present evidence that ABTL0812 impairs (Biotek, Winooski, Vermont, USA). neuroblastoma tumour growth, both in vitro and in vivo through ER stress and UPR activation, leading to cyto- Mouse xenografts toxic autophagy and apoptosis. Furthermore, our results All animal procedures were approved by the Ethics suggest that in neuroblastoma ABTL0812 enhances the Committee for Animal Experimentation of Vall Hebron activity of standard therapies, such as irinotecan and the Research Institute (ref-12/14 CEEA). differentiating agent 13-cis-retinoic acid (13-cRA), thus SH-SY5Y cells were injected in the flank of 6-week-old providing a new therapeutic option for high-risk neuro- female NMRI-Foxn1nu/nu immunodeficient mice (5 × 106 blastoma patients. cells/flank) in 300 μl of PBS and MatrigelTM (Corning, NY, USA) in proportion 1:1. Sample size was calculated Materials and methods using the G*Power software version 3.19.6 (University of Cell lines Kiel, Germany). Power was set to detect differences with P SK-N-AS, SH-SY5Y, and IMR-32 cell lines were pur- value < 0.05 and a 50% difference in tumour growth. Once chased from American Type Culture Collection (ATCC, all mice developed measurable tumours (~70 mm3), mice Manassas, VA, USA), CHLA-90 cell line was acquired were allocated in three different groups by block rando- from the Children’s Oncology Group Cell Culture and misation. The three groups were vehicle (5% glycerol in Xenograft Repository (Lubbock, TX, USA), and SK-N-BE water; n = 10), 120 mg/kg ABTL0812 (n = 10) or 2 mg/kg (2) and LA1-5s were purchased from Public Health Eng- cisplatin (n = 10). ABTL0812 and vehicle were orally land Culture Collections (Salisbury, UK). Neuroblastoma administered 7 days/week. Cisplatin was diluted in PBS cell lines were cultured and maintained in Iscove’s mod- and administered i.p. 2 days/week. Tumour growth and ified Dulbecco’s medium (Thermo Fisher Scientific, mice weight was measured with no blinding using a digital Waltham, MA, USA) supplemented with 10% heat- calliper 2–3 times per week. When primary tumours Official journal of the Cell Death Differentiation Association París-Coderch et al. Cell Death and Disease (2020)11:773 Page 3 of 10 reached 1500 mm3, mice were euthanized and tumours stained chromatin as healthy. Scoring was evaluated by excised and weighted. Only those tumours confirmed by blinded investigators. A minimum of 100 cells were haematoxylin and eosin staining were included in the counted from four representative areas of each well. analyses. Statistical analysis Immunofluorescence Unless otherwise stated, graphs represent the average of Cells were seeded in 24-well plates (8 × 104 cells/well for three independent experiments ± SEM. Statistical sig- SK-N-BE(2); 6 × 104 cells/well for LA1-5s), on slide covers nificance was determined by unpaired two-tailed Stu- coated with 40 μg/ml poly-L-lysine (Sigma-Aldrich). After dent’s t-test or ANOVA Tukey’s test (GraphPad Prism 24 h, cells were treated with the indicated treatments for Software, USA). 12 h. Cells were fixed with 4% paraformaldehyde (Sigma- Aldrich), permeabilized with 0.02% saponin (Sigma- Results Aldrich) and blocked for 1 h with 0.01% saponin, 10 mM ABTL0812 impairs neuroblastoma cell growth in vitro and glycine and 5% BSA (bovine serum albumin; Sigma- tumour growth in vivo Aldrich). Next, cells were incubated overnight with the A panel of neuroblastoma cell lines with clinically LC3 primary antibody diluted in 0.01% saponin and 1% relevant molecular alterations (Supplementary Table 2) BSA in a wet chamber at 4 °C, and then incubated for 1 h was selected to analyse their sensitivity to ABTL0812 or with the secondary antibody. Nuclei were stained with cisplatin, one of the standards-of-care DNA-damaging Hoechst 33342 (Sigma-Aldrich) for 5 min, rinsed and agents used in the induction phase of high-risk neuro- mounted on a slide with a drop of Fluorsave mounting blastoma treatment16. While cisplatin was much more medium (Calbiochem, Darmstadt, Germany). Images effective in neuroblastoma cell lines with functional p53, were captured using a fluorescent microscope (Nikon i.e., SH-SY5Y and IMR-32 (Fig. 1a), ABTL0812 impaired Eclipse 90i, Nikon, Vienna, Austria). the growth of all tested cell lines, regardless of p53 functional status or genomic alterations such as MYCN Western blot amplification (Fig. 1b). IC50 values for ABTL0812 were in Proteins were extracted using RIPA buffer (Thermo the similar range (i.e., 30–60 µM) among all tested cell Fisher Scientific) supplemented with 1× EDTA-free lines. However, IC50 values for cisplatin were at least 10 complete protease inhibitor cocktail (Roche, Basel, Swit- times lower in the p53 wild type cell lines compared to zerland) and the phosphatase inhibitors sodium fluoride p53 mutant cell lines (Supplementary Table 3). and sodium orthovanadate (Sigma-Aldrich). Protein To identify potential adverse effects resulting from concentration was determined using Lowry DC protein exposure to ABTL0812 in children, an Ames test17 was assay (Bio-Rad, Hercules, CA, USA). Cell lysates performed, which showed no mutagenic effect (Supple- (25–30 μg of protein) were resolved on NuPAGETM mentary Fig. 1A, B). Furthermore, in neuroblastoma cells, 4–12% Bis-Tris gels (Thermo Fisher Scientific) and ABTL0812 induced little or no phosphorylation of H2AX transferred into PVDF membranes. Membranes were at serine 139 (a marker of DNA double strand breaks18), blocked for 1 h with 5% non-fat milk or 5% BSA diluted in while p-H2AX was strongly induced by cisplatin (Sup- Tris-buffered saline 0.1% Tween and probed with the plementary Fig. 1C). indicated primary antibodies (see Supplementary Table 1) The impact of ABTL0812 treatment on neuroblastoma overnight at 4 °C. Membranes were incubated with sec- cells was further evaluated in vivo using subcutaneous ondary antibodies for 1 h and developed with chemilu- xenografts (Fig. 1c). While tumours of vehicle-treated minescent horseradish peroxidase substrate Immobilon mice showed a rapid growth, those treated with Western (Millipore, Darmstadt, Germany). Quantifica- ABTL0812 or cisplatin grew at a much slower pace (Fig. tions of western blots were performed with Image J 1d). These effects were confirmed by analysis of tumour (National Institutes of Health, Bethesda, MD, USA). weight at the end of the experiment (Fig. 1e) and validated by a strong correlation between tumour weight and Cell death assays volume (Fig. 1f). Mice weight measurements showed a Cells were seeded in 24-well plates and treated 24 h later slight reduction in body weight in ABTL0812 treated with ABTL0812 or cisplatin. At the end of treatment, cells group (~5%, Supplementary Fig. 2A). We also did a were stained with 0.05 μg/ml Hoechst 33342 (Sigma- complete blood cell count test (CBC) from five to twelve Aldrich) and 2.5 μM propidium iodide (PI, Thermo), and mice from each treatment group. The haematological incubated for 15 min protected from light. Images were analysis showed that ABTL0812 had no impact on hae- captured using a fluorescent microscope (Nikon Eclipse matocrit, haemoglobin and red blood cell counts, neither 90i, Nikon). Cells with condensed or fragmented nuclei in liver and kidney damage indicators AST, ALT and urea were considered as dead cells and those with uniformly concentration (Supplementary Fig. 2B). Official journal of the Cell Death Differentiation Association París-Coderch et al. Cell Death and Disease (2020)11:773 Page 4 of 10 Fig. 1 ABTL0812 reduces neuroblastoma cell lines proliferation in vitro and tumour growth in vivo. a, b Dose-response curves for cisplatin (a) and ABTL0812 (b) in six neuroblastoma cell lines. Cells were treated with ABTL0812 or cisplatin for 72 h at the indicated concentrations, then fixed with 1% glutaraldehyde and stained with crystal violet. c Schematic representation of the in vivo experiment using a neuroblastoma xenograft model. d Tumour volume of mice treated with vehicle, ABTL0812 or cisplatin measured at 23 days (n = 10/group). e Average weight of resected tumours. f Correlation between tumour weight and tumour volume. Data is presented as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01 ABTL0812 vs. vehicle; $ p ≤ 0.05, $$p ≤ 0.01 cisplatin vs. vehicle. Collectively, these data show that ABTL0812 has a immunofluorescence as a hallmark of autophagy. When similar antitumour potential to cisplatin. Conversely, it chemoresistant neuroblastoma cells were treated with does cause neither DNA strand breaks nor mutations. ABTL0812, a remarkable change in LC3 distribution was observed, showing an increase of punctuate/dot LC3-II in ABTL0812 induces cell death in neuroblastoma cell lines both cell lines (Fig. 2a). ABTL0812 is known to impair tumour growth by acti- Cytotoxic autophagy only occurs when the autophagic vation of autophagy-mediated cell death in multiple adult flux is completed and autophagosomes fuse with lyso- cancers11,12,19,20. During autophagy, the cytosolic soluble somes to be degraded, leading to the concomitant loss of protein LC3 is converted to LC3-II, which is bound to LC3-II. Since pre-incubation of cells with lysosomal autophagosomal membranes. Thus, the diffuse ubiquitous degradation inhibitors (E64d and pepstatin A) resulted in LC3 distribution pattern is converted into a punctuate/ increased levels of LC3-II, it can be assumed that dot LC3-II pattern, a process that can be tracked by ABTL0812 induced dynamic autophagic flux (Fig. 2b). Official journal of the Cell Death Differentiation Association París-Coderch et al. Cell Death and Disease (2020)11:773 Page 5 of 10 Fig. 2 ABTL0812 induces autophagy and apoptosis in neuroblastoma cells. a Immunostaining of ABTL0812-induced autophagosome formation. Neuroblastoma cells were treated with 20 µM (SK-N-BE(2)) or 40 µM (LA1-5s) ABTL0812 for 12 h and then fixed and stained with anti-LC3 (green) and DAPI (blue). Punctuated patterns shows LC3-II recruited to the autophagosomes. b Western blot analysis of ABTL0812-induced autophagic flux. Cells were pre-treated 2 h with vehicle or 10 µM E64d and 10 µg/ml pepstatin A (PA). Then, 40 µM (LA1-5s) or 30 µM (SK-N-BE(2)) ABTL0812 was added for 6 h in the presence or absence of lysosomal inhibitors. Anti-TRIB3 was used as a control for ABTL0812 response. c Representative images of nuclear morphology assessment at 48 h post-treatment with ABTL0812 (30 µM for LA1-5s, 20 µM for SK-N-BE(2)) or vehicle. Arrowheads point towards condensed or fragmented nuclei. Cisplatin (25 µM) was used as an apoptosis positive control. d Western blot analysis of ABTL0812-induced apoptosis 72 h post-treatment. e Autophagy and apoptosis inhibitors reduce ABTL0812-induced cell death. LA1-5s and SK-N-BE(2) cells were pre-treated 2 h with vehicle (ethanol) or 10 μM E64d and 10 μg/ml PA or 20 μM QVD. 30 μM ABTL0812 for LA1-5s or 20 μM for SK-N-BE(2) were added in the presence or absence of autophagy and apoptosis inhibitors. Cell death was quantified 48 h post-treatment by scoring four representative fields of each condition (n = 3). Data is presented as the average of three independent experiments ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared to ABTL0812 in the absence of inhibitors. Interestingly, at later time points, Hoechst staining of ABTL0812 treatment triggers autophagy and apoptosis in ABTL0812-treated neuroblastoma cell lines showed the neuroblastoma cell lines. typical morphological features of apoptosis: chromatin condensation, nuclear fragmentation and formation of ABTL0812 induces endoplasmic reticulum stress and the apoptotic bodies (Fig. 2c), thereby suggesting that unfolded protein response ABTL0812 treatment also induced apoptosis in neuro- To shed light on the molecular pathways behind blastoma cells. Indeed, cleavage of caspase-3 and its ABTL0812-induced cell death, we investigated the first substrate PARP was also observed upon ABTL0812 proposed mechanism of action for ABTL0812, the inhi- treatment (Fig. 2d). Moreover, pre-treatment with the bition of the AKT/mTORC1 axis by upregulation of lysosomal protease inhibitors E64d/pepstatin A or with TRIB3 pseudokinase11. Although treatment of neuro- the pan-caspase inhibitor QVD-OPh (QVD) resulted in a blastoma cells with ABTL0812 resulted in increased significant reduction of ABTL0812-induced cell death TRIB3 levels and bi-lipidation of LC3, the levels of AKT (Fig. 2e). Collectively, these results suggest that phosphorylation or its downstream targets PRAS40 and Official journal of the Cell Death Differentiation Association París-Coderch et al. Cell Death and Disease (2020)11:773 Page 6 of 10 Fig. 3 ABTL0812 triggers the UPR-PERK signalling pathway. a Left, representative scheme of the AKT/mTORC1 pathway. Right, western blot of the components of the pathway in LA1-5s and SK-N-BE(2) cell lines treated with the indicated ABTL0812 concentrations for 24 h. b Western blot quantification. Graph represents the mean of two out of three independent experiments. Band intensity was normalised to GAPDH and vehicle- treated cells. *p ≤ 0.05, compared to vehicle. c Left, representative scheme of the UPR PERK pathway. Right, western blot of the components of the pathway in LA1-5s and SK-N-BE(2) cell lines treated with the indicated ABTL0812 concentrations for 0, 3, 6 and 8 h. d Graph represents the mean quantification of two out of three independent experiments. Band intensity was normalised to GAPDH and time 0 h. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared to time = 0. S6 were not modified at the time and doses analysed with these observations, a remarkable upregulation of (Fig. 3a, b). NOXA was seen in both neuroblastoma cell lines analysed Recently, ABTL0812 has also been shown to modulate upon ABTL0812 treatment (Fig. 3c, d). cytotoxic autophagy through induction of ER stress and In summary, these data support the hypothesis that the UPR12. We monitored the impact of ABTL0812 on ABTL0812 induces neuroblastoma cell death through the PERK signalling pathway, a branch of UPR closely induction of ER stress and the PERK branch of the UPR related to ER stress-induced apoptosis21–24. ABTL0812 pathway. treatment resulted in increased phosphorylation of PERK substrate eIF2α, as well as in increased protein levels of ABTL0812 potentiates the antitumoural effect of the downstream effectors ATF4 and the C/EBP homo- chemotherapy and differentiating agents used for the logous protein (CHOP) (Fig. 3c, d). Although the exact management of high risk-neuroblastoma mechanism of how these effectors trigger apoptotic cell Currently, high-risk neuroblastoma is treated with death is not completely understood, they usually do regimens of combined chemotherapies. Hence we aimed through the activation of pro-apoptotic members of the to combine ABTL0812 with the most common drugs used Bcl-2 family such as Bim, NOXA or Puma25. Concurring for patients with high-risk neuroblastoma, such as Official journal of the Cell Death Differentiation Association París-Coderch et al. Cell Death and Disease (2020)11:773 Page 7 of 10 Fig. 4 ABTL0812 potentiates the antitumoural effect of high-risk neuroblastoma treatments. LA1-5s cells were treated with vehicle (ethanol), 30 µM ABTL0812 and the indicated doses of doxorubicin (a), topotecan (b), irinotecan (c) and cyclophosphamide (d) as a single agent (plain bars) or in combination with 30 µM ABTL0812 (striped bars). After 72 h of treatment, cells were fixed with 1% glutaraldehyde and proliferation was assessed by crystal violet staining. Data is presented as the average of three independent experiments ± SEM. *, #, $ mean p ≤ 0.05, **p ≤ 0.01. “*” indicates comparison vs. vehicle; “#” compares vs. ABTL0812 as a single agent; and “$” compares vs. the indicated drug as single agent. doxorubicin, irinotecan, topotecan and cyclopho- Concurring with this evidence, the apoptotic response was sphamide. Three different doses of each compound, alone observed only when 13-cRA and ABTL0812 were added or in combination with 30 µM ABTL0812 were tested together. In both cell lines, the combined treatment (Fig. 4a-d). While a positive trend was observed for all induced the cleavage of caspase-3 and its substrate PARP- compounds, the strongest antitumoural effect was seen 1, confirming activation of apoptosis (Fig. 5b). when ABTL0812 was combined with irinotecan. At the In conclusion, these results suggest that ABTL0812 three tested concentrations, the addition of irinotecan potentiates the antitumoural effect of high-risk neuro- increased the cytotoxic effect of ABTL0812 (Fig. 4c). blastoma chemotherapy drugs and differentiating agents Finally, we investigated the effect of ABTL0812 com- used for the treatment of minimal residual disease. bined with biologic agents used for the treatment of minimal residual disease (MRD) in neuroblastoma, such Discussion as 13-cis-retinoic acid (13-cRA). Cell proliferation assays ABTL0812 is a first-in-class chemically modified poly- were performed in LA1-5s and SK-N-BE(2) neuro- unsaturated fatty acid which has demonstrated anti- blastoma cell lines treated simultaneously with ABTL0812 tumour activity in several in vitro and in vivo models of and three doses of 13-cRA. Administration of low 13-cRA adult cancers11,19,20. Our results show that ABTL0812 doses as single agent had little effect on neuroblastoma impairs neuroblastoma viability regardless of poor prog- cell proliferation at 24 h post-treatment; however, the nosis markers, such as MYCN amplification and p53 combination of 13-cRA with ABTL0812 induced a strong mutations. Unlike what it has been described for lung, reduction in the viability of both neuroblastoma cell lines pancreatic and endometrial cancer cells (6,10,11), (Fig. 5a). Interestingly, both drugs, ABTL0812 and 13- ABTL0812 does not reduce phosphorylation of AKT or cRA, caused an upregulation of ABTL0812 effectors such mTORC1 substrates, despite inducing the upregulation of as TRIB3, ATF4 and bi-lipidation of LC3, which were TRIB3 and LC3-II levels. Thus, autophagy-mediated cell further enhanced when both drugs were combined. death in neuroblastoma is independent of the AKT/ Official journal of the Cell Death Differentiation Association París-Coderch et al. Cell Death and Disease (2020)11:773 Page 8 of 10 Fig. 5 ABTL0812 potentiates the antitumoural effect of 13-cis-retinoic acid. LA1-5s and SK-N-BE(2) cells were treated with vehicle (ethanol), 20 µM ABTL0812 or the indicated doses of 13-cRA, alone or in combination with ABTL0812. a Cell proliferation assay. Neuroblastoma cells were treated for 24 h and then fixed with 1% glutaraldehyde and stained with crystal violet. Graph represents the average of three independent experiments ± SEM. Plain bars represent single agent treatments and striped bars combined treatments. *, #, $p ≤ 0.05, **, $$p ≤ 0.01, ***, ###p ≤ 0.001. “*” indicates comparison vs. vehicle; “#” compares vs. ABTL0812 as a single agent; and “$” compares vs. 13-cRA as single agent. b Western blot of the indicated proteins after treatment with both vehicles, 20 µM of ABTL0812, 20 µM of 13-cRA or the combination, for 24 h. mTOR axis. In addition, we observed that neuroblastoma ABTL0812 as a potential therapeutic option for neuro- cells died by apoptosis, in accordance to what was observed blastoma patients with a higher safety profile than current for endometrial cancer models (10). Concurring with recent treatments. This preclinical data supported the findings12, our results suggest that ABTL0812 induces ABTL0812 Orphan Drug Designation (ODD) by the FDA prolonged ER stress and activation of the PERK/ATF4/ (15‐4893) and EMA (EU/3/15/1485) agencies for CHOP/NOXA UPR branch. This novel mechanism of neuroblastoma. action would help to overcome some of the resistance ABTL0812 has also shown synergistic effect in adult mechanisms present in high-risk neuroblastoma tumours, tumours when combined with standard che- such as defective p53 signalling and caspase 8 silencing26–28. motherapies19,20. Therefore, we analysed the potential Cumulative preclinical data and the first-in-human benefit of combining ABTL0812 with chemotherapies phase I/Ib clinical trial of ABTL0812 as a single therapy used to treat high-risk neuroblastoma, either in first or (NCT02201823) showed high safety and tolerability. In second line, such as irinotecan, topotecan, doxorubicin fact, the maximum tolerated dose was not achieved and cyclophosphamide. Although our results suggest that because no dose‐limiting toxicities were identi- ABTL0812 enhanced the therapeutic effect of these drugs, fied11,15,19,20. Accordingly, our results confirmed that the potentiation effect was modest. However, we did see a ABTL0812 is not mutagenic and does not induce DNA remarkable effect when we combined ABTL0812 with damage. Nonetheless, the in vivo efficacy of ABTL0812 is 13c-RA, one of the biological agents used for the treat- similar to cisplatin, a DNA-damaging chemotherapy drug ment of minimal residual disease in neuroblastoma29,30. used for neuroblastoma treatment. These results promote Our molecular assays showed that low doses of 13-cRA Official journal of the Cell Death Differentiation Association París-Coderch et al. Cell Death and Disease (2020)11:773 Page 9 of 10 were activating the PERK/ATF4/CHOP axis and autop- References hagy, but not cell death. It has been reported that cell 1. Maris, J. M. Recent advances in neuroblastoma. N. Engl. J. Med. 362, 2202–2211 (2010). death only occurs through the UPR when the ER stress 2. Matthay, K. K. et al. Neuroblastoma. Nat. Rev. Dis. Prim. 2, 16078 (2016). stimuli is intense and prolonged (reviewed in ref. 31). 3. Maris, J. M., Hogarty, M. D., Bagatell, R. & Cohn, S. L. Neuroblastoma. 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Honokiol induces autophagy of neuroblastoma cells through ABTL0812 is used as a single agent. activating the PI3K/Akt/mTOR and endoplasmic reticular stress/ERK1/2 signal- In summary, our results strengthen the notion that ing pathways and suppressing cell migration. Cancer Lett. 370, 66–77 (2016). prolonged and intense ER stress and UPR induction by 9. Bastola, T., An, R. B., Kim, Y. C., Kim, J. & Seo, J. Cearoin induces autophagy, ERK activation and apoptosis via ROS generation in SH-SY5Y neuroblastoma cells. ABTL0812 offers a new, safe and effective anti-cancer Molecules 22, E242 (2017). therapeutic strategy for patients with high-risk 10. Han, Z. et al. Norcantharidin inhibits SK-N-SH neuroblastoma cell growth by neuroblastoma. induction of autophagy and apoptosis. Technol. Cancer Res. Treat. 16, 33–44 (2017). 11. Erazo, T. et al. The new antitumor drug ABTL0812 inhibits the Akt/mTORC1 Acknowledgements axis by upregulating tribbles-3 pseudokinase. Clin. Cancer Res. 22, 2508–2519 We thank Ms. Christin O’Hara for text revision. This work was funded by (2016). Instituto de Salud Carlos III (Grant no. CP16/00006, PI17/00564 to M.F Segura); 12. Muñoz-Guardiola, P. et al. The anticancer drug ABTL0812 induces ER stress- Ministerio de Educación, Cultura y Deporte (Grant no. FJCI_2015_24212 to R mediated cytotoxic autophagy by increasing dihydroceramide levels in cancer Antonelli; Grant no. FPU16/01099 to M Masanas); Generalitat de Catalunya cells. Autophagy 25, 1–18 (2020). AGAUR/European Social Fund (Grant no. 2017FI_B_00095 to C Jiménez); 13. Fulda, S. The PI3K/Akt/mTOR pathway as therapeutic target in neuroblastoma. Asociación Acunapatata, Asociación NEN. Work in Lizcano’s laboratory was Curr. Cancer Drug Targets 9, 729–737 (2009). supported by grants from the Spanish Ministry of Economy and 14. Vaughan, L. et al. Inhibition of mTOR-kinase destabilizes MYCN and is a Competitiveness (MINECO) INNPACTO/IPT-2012-0614-010000 and SAF2015- potential therapy for MYCN-dependent tumors. Oncotarget 7, 57525–57544 64237-R, and co-funded by the European Regional Development Fund (ERDF). (2016). 15. Vidal Boixader, L. et al. Dose-escalation of the first-in human phase I/Ib study Author details of ABTL0812, a novel antitumor drug inhibiting the Akt/mTOR pathway in 1 Translational Research in Child and Adolescent Cancer, Vall d’Hebron Institut patients with advanced solid tumors. J. Clin. Oncol. 33, 2585 (2015). de Recerca (VHIR), Barcelona, Spain. 2Protein Kinases and Signal Transduction 16. Ganeshan, V. R. & Schor, N. F. Pharmacologic management of high-risk neu- Laboratory, Institut de Neurociències and Departament de Bioquímica i roblastoma in children. Pediatr. Drugs 13, 245–255 (2011). Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Spain. 17. Ames, B. N., McCann, J. & Yamasaki, E. 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