Alejandro Losada

Aplidin ™ induces the mitochondrial apoptotic pathway via oxidative stress-mediated JNK and p38 activation and protein kinase C δ

Aplidin™, a new antitumoural drug presently in phase II clinical trials, has shown both in vitro and in vivo activity against human cancer cells. Aplidin™ effectively inhibits cell viability by triggering a canonical apoptotic program resulting in alterations in cell morphology, caspase activation, and chromatin fragmentation. Pro-apoptotic concentrations of Aplidin™ induce early oxidative stress, which results in a rapid and persistent activation of both JNK and p38 MAPK and a biphasic activation of ERK. Inhibition of JNK and p38 MAPK blocks the apoptotic program induced by Aplidin™, demonstrating its central role in the integration of the cellular stress induced by the drug. JNK and p38 MAPK activation results in downstream cytochrome c release and activation of caspases -9 and -3 and PARP cleavage, demonstrating the mediation of the mitochondrial apoptotic pathway in this process. We also demonstrate that protein kinase C delta (PKC-δ) mediates the cytotoxic effect of Aplidin™ and that it is concomitantly processed and activated late in the apoptotic process by a caspase mediated mechanism. Remarkably, cells deficient in PKC-δ show enhanced survival upon drug treatment as compared to its wild type counterpart. PKC-δ thus appears as an important component necessary for full caspase cascade activation and execution of apoptosis, which most probably initiates a positive feedback loop further amplifying the apoptotic process.

Lung hypoplasia caused by nitrofen is mediated by down-regulation of thyroid transcription factor TTF-1

Abstract Prenatal exposure to nitrofen induces lung hypoplasia and diaphragmatic hernias very similar to those in human disease, but the mechanisms are still unknown. Thyroid transcription factor 1 (TTF-1) is involved in lung ontogeny and regulation of the expression of surfactant proteins, and is likely abnormally expressed in nitrofen-induced lung hypoplasia. This study examines the effect of nitrofen on TTF-1 messenger RNA (mRNA) expression in the lungs of prenatal rat fetuses and a human lung-cell line (NCI-H441) that expresses both TTF-1 and surfactant proteins in vivo. Lungs from preterm fetuses harvested from rats with 100 mg nitrofen on gestational day 9.5 and NCI-H441 cells maintained in RPMI medium containing 10% fetal bovine serum and exposed to nitrofen for different times and concentrations were assayed for TTF-1 mRNA by northern blot analysis. mRNA for TTF-1 was decreased in nitrofen-exposed pups in comparison with controls, and exposure to nitrofen caused a dose- and time-related decrease in TTF-1 expression in H441 cell cultures. These results indicate that nitrofen downregulates TTF-1 both in vivo and in vitro. Since this interferes with lung development, it is reasonable to accept that lung hypoplasia in this model is in part due to the direct effect of the teratogen rather than to compression by the abdominal viscera herniated into the thorax. This mechanism should be explored in the clinical setting.

Abstract 5430: eEF1A2 is a new target for anticancer therapy

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Eukaryotic Elongation Factor 1A2 (eEF1A2) is an isoform of the alpha subunit of eEF1 complex. Differently from the A1 isoform, the expression of eEF1A2 is restricted to brain, heart and skeletal muscle. eEF1A2 is overexpressed in tumors, including multiple myeloma (MM) (Li et al, 2012, PLOS One 5, e10755), prostate (Sun et al, 2014, Biochem Biophys Res Commun 450:1-6), pancreas (Xu et al, 2013, Clin Exp Metastasis 30:933-44) and ovarian (Anand et al, 2002, Nat Genet 31:301-5), and has also an oncogenic behavior favoring tumor cell proliferation while inhibiting apoptosis (Lee and Surh, 2009, Ann N Y Acad Sci 1171:87-93). Thus, eEF1A2 is an interesting target for cancer treatment. Aplidin (plitidepsin) is an antitumor agent, originally isolated from the marine tunicate Aplidium albicans, which is being tested in MM patients in a phase III pivotal trial in combination with dexamethasone and a phase I trial in combination with bortezomib and dexamethasone. Herein we reveal the interaction of Aplidin with eEF1A2 using different methods; i) a DARTS assay showed that Aplidin protected eEF1A2 from digestion by the protease subtilisin (EC 3.4.21.62); ii) a saturation binding experiment using [14C]-Aplidin and eEF1A2 purified from rabbit muscle determined a Kd for the interaction of approximately 80 nM; iii) a fluorescence anisotropy test through two photon microscopy showed that Aplidin preferentially binds the GTP-bound form of eEF1A2. Furthermore, we performed a [14C]-Aplidin binding-guided fractionation of K562 cell extracts through differential centrifugation and several chromatographic steps, including ion-exchange and size-exclusion chromatography, demonstrating that eEF1A2 is the unique cellular protein that can be retrieved through specific binding to Aplidin. In addition, HeLa-APL-R cells, a HeLa subclone made extremely resistant to Aplidin, more than 1000 fold, (Losada et al., 2004, Br J Cancer 91:1405-13), are shown to express 8 fold less eEF1A2 than the parental cells both at the mRNA (determined with an Affimetryx HG-U133A Array) and protein (determined by iTRAQ) levels. When normal eEF1A2 levels were restored in HeLa-APL-R cells through ectopic expression of an eEF1A2-GFP construct, these cells were rendered partially sensitive to Aplidin. Interestingly, the transfected cells recovered most of the signaling events which are typically induced by the drug in HeLa wt cells. Altogether, our results demonstrate that eEF1A2, the oncogenic isoform of the alpha subunit of eEF1, is the primary target of Aplidin and a new suitable and druggable target for anticancer therapy. Citation Format: Alejandro Losada, Maria Jose Munoz, Carolina Garcia, Juan F. Martinez-Leal, Federico Gago, Carmen Cuevas, Luis F. Garcia-Fernandez, Juan M. Dominguez, Pilar Lillo, Carlos M. Galmarini. eEF1A2 is a new target for anticancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5430. doi:10.1158/1538-7445.AM2015-5430

Dynamic cellular maps of molecular species: Application to drug-target interactions

The design of living cell studies aimed at deciphering the mechanism of action of drugs targeting proteins with multiple functions, expressed in a wide range of concentrations and cellular locations, is a real challenge. We recently showed that the antitumor drug plitidepsin (APL) localizes sufficiently close to the elongation factor eEF1A2 so as to suggest the formation of drug-protein complexes in living cells. Here we present an extension of our previous micro-spectroscopy study, that combines Generalized Polarization (GP) images, with the phasor approach and fluorescence lifetime imaging microscopy (FLIM), using a 7-aminocoumarin drug analog (APL*) as fluorescence tracer. Using the proposed methodology, we were able to follow in real time the formation and relative distribution of two sets of APL-target complexes in live cells, revealing two distinct patterns of behavior for HeLa-wt and APL resistant HeLa-APL-R cells. The information obtained may complement and facilitate the design of new experiments and the global interpretation of the results obtained with other biochemical and cell biology methods, as well as possibly opening new avenues of study to decipher the mechanism of action of new drugs.

Abstract 2129: Aplidin triggers the activation of molecular components of the UPR as part of its pro-apoptotic program in tumor cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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. Martinez-Leal, Alberto Bejarano, Carmen Cuevas, Luis F. Garcia-Fernandez, 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

Abstract 1165: Plitidepsin targets the moonlighting functions of eEF1A2 in cancer cells

Plitidepsin, a cyclic depsipeptide of marine origin, has shown potent anticancer activity in preclinical assays and recently finished with positive results a pivotal phase III trial (clinicaltrials.gov identifier: NCT01102426) for the treatment of multiple myeloma patients. We have recently found that eukaryotic elongation factor 1A2 (eEF1A2), one of the two isoforms of the alpha subunit of eEF1, is the pharmacological target of plitidepsin. Although it shares 96% homology with eEF1A1 (the other isoform), they display an exclusive pattern of expression, being eEF1A2 solely expressed in brain and muscle in healthy individuals. However, it has been found that many tumors abnormally overexpress this protein, including multiple myeloma, prostate, pancreatic, ovarian, breast, lung and liver cancers. Furthermore, although eEF1A2 canonical function consists in the delivery of aminoacyl-tRNAs to the A site in the ribosome, it has been shown to have pro-oncogenic moonlighting activities, including inhibition of apoptosis, protein degradation by the proteasome, heat shock response, cytoskeleton organization and regulation of oxidative stress. We now investigated several of the pro-oncogenic activities of eEF1A2 to analyze the impact that plitidepsin could have preventing them. Indeed, we observed that plitidepsin interfered with the interaction between eEF1A2 and Peroxiredoxin 1 (PRDX1), a complex that allosterically enhances the enzymatic activity of PRDX1. This way, plitidepsin would diminish PRDX1 antioxidant activity, possibly originating the oxidative stress that has been described in the bibliography as one of the first effects triggered by the drug in cancer cells. PRDX1 only interacts with the GDP-bound form of eEF1A2, while plitidepsin exclusively binds to the GTP-bound form, most probably sequestering this protein from the pool that could interact with and activate PRDX1. Furthermore, we have confirmed that eEF1A2 interacts with Sphingosine kinase 1 (SPHK1), a complex that has been described in the bibliography as having enhanced SPHK1 activity. SPHK1 phosphorylates sphingosine producing sphingosine-1-P, second messenger that binds to its receptors in the cell membrane and conveys growth and survival signals to the cell. We could see that plitidepsin treatment reduced the production of sphingosine-1-P in HeLa cells, destabilizing the equilibrium towards the pro-apoptotic ceramide/sphingosine side and promoting cell death. Thus, through its binding to eEF1A2, plitidepsin derails a series of its moonlighting functions that are essential for the survival of tumor cells, driving them into apoptosis. Citation Format: Alejandro Losada, Maria Jose Munoz, Juan F. Martinez-Leal, Juan M. Dominguez, Carlos M. Galmarini. Plitidepsin targets the moonlighting functions of eEF1A2 in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1165. doi:10.1158/1538-7445.AM2017-1165

Abstract 3015: Plitidepsin targets the GTP-bound form of eEF1A2 in cancer cells

Plitidepsin (APL), an antitumor agent originally isolated from the marine tunicate Aplidium albicans, is being tested in multiple myeloma (MM) patients in a phase III pivotal trial in combination with dexamethasone and a phase I trial in combination with bortezomib and dexamethasone. eEF1A2 is 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 pro-oncogenic activities (5). Here we analyze the interaction of plitidepsin with its target, eEF1A2. DARTS assays, either with whole cell extracts or with purified eEF1A2 protein, indicate that plitidepsin binds to eEF1A2 and protects it from digestion by subtilisin (EC 3.4.21.62). Moreover, a [14C]-APL binding-guided fractionation of K562 cell extracts through differential centrifugation and three chromatographic steps demonstrated that eEF1A2 is the only cellular protein that can be retrieved through specific binding to plitidepsin. A saturation binding experiment with [14C]-APL and purified GTP-bound eEF1A2 (from rabbit muscle) allowed us to calculate a Kd of around 80 nM for the interaction, while a dissociation experiment revealed a residence time of around 9 minutes. Indeed, we have found that plitidepsin exclusively binds to the GTP-bound form of eEF1A2. HeLa-APL-R cells, ≥1000 fold more resistant to plitidepsin than parental HeLa wt cells (6), are now shown to have lower eEF1A2 mRNA and protein levels than parental cells. Furthermore, when eEF1A2 levels were restored to normal in HeLa-APL-R cells through ectopic expression of an eEF1A2-GFP construct, they were rendered partially sensitive to plitidepsin. Interestingly, transfected cells recovered most of the signaling events typically induced by the drug in HeLa wt cells. NCI-H460 (lung) and HGC-27 (stomach) cancer cell lines were rendered resistant to plitidepsin following the same procedure described in (6) for HeLa-APL-R cells. When we analyzed the levels of eEF1A2 in this two new cell lines we observed that both of them were lacking eEF1A2. Altogether, our results demonstrate that plitidepsin targets the pro-oncogenic eEF1A2 protein, a new druggable target for anticancer therapy. (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 Citation Format: Alejandro Losada, Maria J. Munoz-Alonso, Juan F. Martinez-Leal, Juan M. Dominguez, Carlos M. Galmarini. Plitidepsin targets the GTP-bound form of eEF1A2 in cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3015.

Abstract 5467: Role of the eukaryotic elongation factor eEF1A in the mechanism of action of Aplidin

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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 didemnins 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. Martinez-Leal, Federico Gago, Carmen Cuevas, Luis F. Garcia-Fernandez, 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

Abstract 4655: Aplidin induces a non-canonical ER stress response in HeLa cells response in HeLa cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Aplidin (APL), a marine cyclic depsipeptide originally found in the Mediterranean tunicate Aplidium albicans, is currently under phase II/III clinical investigation for cancer therapy. The mechanism of action of the compound includes the oxidative-stress mediated activation of Rac1 and JNK1, which rapidly trigger the mitochondrial apoptotic pathway. Comparing the protein expression profiles of HeLa wt cells and an APL-resistant subclone previously generated in our lab (HeLa-APL), we identified a subset of differentially expressed proteins the function of which was related to the unfolded protein response (UPR). HeLa-APL resistant cells showed reduced levels of BiP (GRP78) and relatively higher levels of Ero1α and phospho-eIF2α, indicating a higher basal endoplasmic reticulum (ER) stress. To investigate whether APL was inducing a bona fide ER stress response in HeLa cells and whether this process was essential in the mechanism of action of this compound, we compared the molecular and cellular effects elicited by APL with those induced by two well-known ER-stress inducing agents, thapsigargin and tunicamycin. Basically, while these agents elicited a complete canonical UPR response, APL only triggered part of the stress signaling cascade, including the phosphorylation of eIF2α and JNK1 and a rapid inhibition of protein synthesis. By contrast, CHOP, a transcription factor involved in launching apoptosis by the UPR, was not induced by APL. Rather, it seemed to be slightly reduced in treated cells. Similarly, while tunicamycin induced the alternative splicing of XBP1 by IRE1 and the activation of the ER-related caspase-4, APL failed to induce the same response in HeLa cells. At present, the precise connection between the partial activation of the ER stress signaling pathway and the rapid induction of apoptosis by APL remains poorly understood, although it could represent a new, aberrant UPR response Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4655. doi:1538-7445.AM2012-4655