Plitidepsin Inhibits Autophagy, The Main Mechanism Of Acquired Resistance To Bortezomib

Background: Plitidepsin (APL), an antitumor agent isolated from the marine tunicate Aplidium albicans, has been tested with positive results in multiple myeloma (MM) patients in a phase III pivotal trial in combination with dexamethasone (clinicaltrials.gov identifier: NCT01102426) and in a phase I trial in combination with bortezomib and dexamethasone (clinicaltrials.gov identifier: NCT02100657). Plitidepsin targets eEF1A2, one of two isoforms of the alpha subunit of the eEF1 complex. In mammals, eEF1A2 has a selective pattern of expression in those tissues that do not express the A1 isoform, namely brain and muscle. Nonetheless, eEF1A2 is aberrantly expressed in many cancers, including solid tumors (1-3) and MM (4), and has been shown to hold oncogenic properties (5). We have previously demonstrated through several means the interaction of plitidepsin with its target, eEF1A2 and calculated a Kd of around 80 nM for this interaction (6). Indeed, we have found that plitidepsin exclusively binds to the GTP-bound form of eEF1A2 Aims: Here we investigated whether eEF1A2 levels had any effect on the sensitivity of MM cells to both plitidepsin and bortezomib, to better understand the synergistic effect these two drugs yield when combined (7). Methods: RPMI-8226, OPM1, U266-B1, MM1S and NCI-NH929 multiple myeloma cells were cultured in optimal conditions. eEF1A2 (GTX102326) and autophagy (L7543) were followed by Western blot. Dose-response experiments were performed in 96 well plates, treating cells with increasing doses of the compounds for 24 hours and measuring cell growth with MTT. Curves were then fitted with GraphPad Prism5 software. For combination experiments, the Chou-Talalay procedure was used. Results: We first analyzed the level of eEF1A2 protein in a panel of multiple myeloma cell lines. Then, we selected a cell line with low expression, MM-1S, and one with high expression, OPM-1, of the elongation factor and checked the sensitivity to both, bortezomib and plitidepsin through dose-response experiments. Interestingly, the overexpression of eEF1A2, the target of plitidepsin, seems to be related to an increased resistance to bortezomib, a phenomenon usually related to enhanced autophagy since this latter pathway compensates the inhibition of proteasome. eEF1A, in its GTP-bound form, has been shown to inhibit chaperone-mediated autophagy (CMA), essential for the degradation of unfolded proteins (8). Since plitidepsin binds to the GTP-bound eEF1A2, probably stabilizing this form of the elongation factor, it seems likely that, in such a way, it may inhibit CMA. We explored this possibility and, indeed, we observed that plitidepsin inhibited autophagy in MM cells. Summary/Conclusion: Plitidepsin treatment presumably counteracts the main mechanism of acquired resistance to bortezomib, namely autophagy. (1)Sun et al., 2014, Biochem Biophys Res Commun 450:1-6. (2)Xu et al., 2013, Clin Exp Metastasis 30:933-44. (3)Anand et al., 2002, Nat Genet 31:301-5. (4)Li et al., 2010, PLOS One 5, e10755. (5)Lee and Surh, 2009, Ann N Y Acad Sci 1171:87-93. (6)Losada et al., 2004, Br J Cancer 91:1405-13. (7)Mitsiades et al., 2008, Cancer Res 68:5216-25. (8)Bandyopadhyay et al., 2010, Mol Cel 39:535-47. Citation Format: Juan F. Martinez-Leal, Gaelle Quiniou, Alejandro Losada, Maria Jose Munoz, Rafael Sanchez-Mesa, Patricia Martinez-Rivas, Juan Manuel Dominguez, Carlos M. Galmarini. Plitidepsin inhibits autophagy, the main mechanism of acquired resistance to bortezomib [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B057.