Abstract 5467: Role of the eukaryotic elongation factor eEF1A in the mechanism of action of Aplidin
Authors: Losada Alejandro , Martínez-Leal Juan F. , Gago Federico , Cuevas Carmen , Garcia-Fernández Luis F. , Galmarini Carlos M. 
Published: May 2015

Abstract
Aplidin is a cyclic depsipeptide of the family of didemnins, originally isolated from the colonial tunicate Aplidium albicans. Aplidin is being evaluated in a phase III clinical trial in patients with relapse or refractory multiple myeloma. As part of its antitumor activity, Aplidin induces rapid oxidative stress, activation of Rac1 and phosphorylation of p38 and JNK1 stress kinases, which together trigger the apoptotic death of tumor cells. Didemnin B (DB), a molecule closely related to Aplidin, has been previously shown to interact with the GTP-bound conformation of the eukaryotic elongation factor eEF1A, an interaction that was related to the didemnin's B ability to inhibit protein synthesis (J. Biol. Chem. 1994, 269:15411-14). A structural model for this interaction has been proposed (J. Med. Chem. 2004, 47:4439-52). We wanted to investigate whether eEF1A had any role in the mechanism of action of Aplidin. Using the DARTS technique, we observed that Aplidin treatment of tumor cells and subsequent digestion of the cellular extracts with different proteases, resulted in a significant increase in the stabililty of eEF1A against protease digestion, suggesting a direct effect of Aplidin on this protein. We previously generated, by continuous exposure to increasing concentrations of the drug, a HeLa derivative cell line (HeLa-APL-R) that showed specific resistance to Aplidin as well as to other related didemnins and tamandarins (Br. J. Cancer 2004, 91:1405-13). We investigated whether there was any difference in the expression levels of eEF1A between HeLa-wt and HeLa-APL-R cell lines. Since two isoforms of the elongation factor are expressed in tumor cells, eEF1A1 and eEF1A2, we checked the relative amount of both proteins at the mRNA and protein levels using DNA microarrays and iTRAQ, respectively. Remarkably, we observed that the mRNA and protein levels of eEF1A2 isoform were lower in HeLa-APL-R resistant cells as compared to their parental cell line. No significant changes were seen in the levels of eEF1A1. The reduced levels of eEF1A2 protein in HeLa-APL-R cells were further confirmed by western blotting using isoform-specific antibodies. To explore the effect of the restoration of the eEF1A2 levels in the HeLa resistant clone, we generated two cell lines stably overexpressing eEF1A1 or eEF1A2 and checked their sensitivity to Aplidin in dose-response cytotoxicity experiments. Both cell lines partially recovered the sensitivity to Aplidin, with the eEF1A2-overexpressing cell line having an even slightly higher sensitivity to the compound. In eEF1A overexpressing cells, Aplidin induced a robust cytostatic effect. At the molecular level, Aplidin induced the phosphorylation of p38 as well as ERK MAPKs, but not JNK phosphorylation or PARP cleavage, two key events in the cytotoxic signaling of the drug. These results could indicate a role of eEF1A in the biological activity of Aplidin in tumor cells.
Citation Format: Alejandro Losada, Juan F. Martínez-Leal, Federico Gago, Carmen Cuevas, Luis F. Garcia-Fernández, Carlos M. Galmarini. Role of the eukaryotic elongation factor eEF1A in the mechanism of action of Aplidin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5467. doi:10.1158/1538-7445.AM2014-5467