Abstract Aplidin (APL), a cyclic depsipeptide originally isolated from the marine tunicate Aplidium albicans and currently under phase II/III clinical trials for cancer therapy, induces oxidative stress, activation of Rac1 and phosphorylation of JNK1, which together trigger a rapid apoptotic program in tumor cells. A previously generated APL-resistant HeLa cell line (APL-R) allowed us to find out differences in protein expression with the parental HeLa cells. Among the proteins differentially expressed, several endoplasmic reticulum (ER) stress-related proteins were observed. We found decreased basal levels of BiP in HeLa APL-R, while the levels of Ero1a and phospho-eIF2a were slightly increased when compared to parental HeLa cells, indicative of a higher basal ER stress. In this work, we investigated if APL was inducing a bona fide ER stress in HeLa cells and whether this process was essential in the mechanism of action of the compound. Similarly to that observed with thapsigargin and tunicamycin, two well known ER stress inducing agents, APL triggered the activation of several key molecular components of a classical ER stress induced unfolded protein response (UPR), including the phosphorylation of eIF2a and JNK1, the proteolitic processing of ATF6 and the alternative splicing of XBP1, but not others, such as the accumulation of noxa or the proteolitic activation of caspase 4. Interestingly, although the eIF2a phosphorylation induced by APL in HeLa wt cells correlated with a strong inhibition of protein synthesis, it did not elicit an increased expression of CHOP, a transcription factor involved in the launch of UPR mediated apoptosis. Actually, it was observed a clear reduction of CHOP protein levels after the treatment of HeLa cells with APL, most probably due to its rapid degradation by the proteasome machinery. In summary, as a part of the proapoptotic program triggered by APL in HeLa cells, we observed the induction of ER stress and the activation of several endpoint executioners of the UPR apoptotic branch. Citation Format: Alejandro Losada, Juan F. Martínez-Leal, Alberto Bejarano, Carmen Cuevas, Luis F. Garcia-Fernández, Carlos M. Galmarini. Aplidin triggers the activation of molecular components of the UPR as part of its pro-apoptotic program in tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2129. doi:10.1158/1538-7445.AM2013-2129