The antitumour drug ABTL0812 impairs neuroblastoma growth through endoplasmic reticulum stress-mediated autophagy and apoptosis

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 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.

Conflict of interest statement

P.M., H.P.M., M.Y.V., J.A. and C.D. are Ability Pharmaceuticals employees; C.D. holds shares of the company; J.M.L., M.F.S. and S.G. are advisory members of Ability Pharmaceuticals. The remaining authors declare that they have no conflict of interest.

Figures

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.

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.

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.

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.

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.