Search

There is a great need to develop novel approaches to target oncogenic transcription factors with small molecules. Ewing sarcoma is emblematic of this need, as it depends on the continued activity of the EWS-FLI1 transcription factor to maintain the malignant phenotype. We have previously shown that the small molecule trabectedin interferes with EWS-FLI1. Here, we report important mechanistic advances and a second-generation inhibitor to provide insight into the therapeutic targeting of EWS-FLI1. We discovered that trabectedin functionally inactivated EWS-FLI1 by redistributing the protein within the nucleus to the nucleolus. This effect was rooted in the wild-type functions of the EWSR1, compromising the N-terminal half of the chimeric oncoprotein, which is known to be similarly redistributed within the nucleus in the presence of UV light damage. A second-generation trabectedin analogue lurbinectedin (PM01183) caused the same nuclear redistribution of EWS-FLI1, leading to a loss of activity at the promoter, mRNA, and protein levels of expression. Tumor xenograft studies confirmed this effect, and it was increased in combination with irinotecan, leading to tumor regression and replacement of Ewing sarcoma cells with benign fat cells. The net result of combined lurbinectedin and irinotecan treatment was a complete reversal of EWS-FLI1 activity and elimination of established tumors in 30% to 70% of mice after only 11 days of therapy. Our results illustrate the preclinical safety and efficacy of a disease-specific therapy targeting the central oncogenic driver in Ewing sarcoma. Cancer Res; 76(22); 6657–68. ©2016 AACR.

Purpose of the Study: Marine sponges have developed mechanisms to protect themselves from a hostile marine microenvironment. One of these mechanisms is the use of biologically active metabolites, explored nowadays for their anticancer properties. PM060184 is one of these compounds, isolated from the Madagascan sponge Lithoplocamia lithistoides. This polyketide is currently under evaluation on clinical trials, and its antitumor activity was previously reported in the preclinical setting in a panel of cell lines and subcutaneous tumor xenografts of different origin. The purpose of the study is to explore the effect of PM060184 in a panel of ovarian high-grade serous (HGS) carcinoma cell lines with different sensitivity to cisplatin and paclitaxel. Experimental Procedures: Cell growth inhibition was analyzed by exposure of increasing concentrations of PM060184 in 96 multiwell plates for 72h, by subsequent confluence evaluation and sulphorhodamine (SRB) staining. Cell cycle experiments were done by propidium iodide (PI) staining after treatment of cell lines at their IC50 value for 72h. Three independent experiments with 6 replicates per condition were performed for both approaches. Celigo Image Cytometer platform was employed for phase contrast and viable cells discrimination with a triple staining (Hoechst, PI and calcein AM), as well as for cell cycle analyses. Results: We have focused on the effect of this drug on a panel of HGS ovarian carcinoma cell lines, previously characterized by their response to cisplatin and paclitaxel. We have observed antiproliferative activity in a concentration-dependent manner, with IC50 values at subnanomolar concentrations in all the cell lines tested. This effect was observed in platinum-sensitive and -resistant cell lines and all of them were also more sensitive to PM060184 than paclitaxel, another tubulin-binding agent usually used for the treatment of ovarian cancer. We have evaluated cell cycle and apoptosis at the IC50 values for each cell line. We have seen that this agent disrupts the cell cycle at different phases, as DNA synthesis and mitosis, depending on the cell line. Additionally, an increase in apoptotic cell death is detected as a sub G1 peak by flow cytometry in most of the cell lines tested. Conclusions: PM060184 is a new tubulin-binding agent with a potent antitumor activity in a panel of HGS ovarian cancer cell lines, with different ranges of sensitivity to cisplatin and paclitaxel. Additionally, this effect is more potent than that exerted by other tubulin-binding compounds, such as paclitaxel. However, the underlying mechanism of PM060184 action on ovarian HGS cell lines will need to be further elucidated. Citation Format: Victoria Heredia-Soto, Andrés Redondo, Alejandro Gallego, María Miguel-Martín, Roberto Crespo, Alicia Hernández, David Hardisson, Jaime Feliu, Carlos Galmarini, Marta Mendiola. Exploratory testing of PM060184 compound in high-grade serous ovarian carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5876.

Human translation elongation factor 1α2, encoded by the eEF1A2 gene, is a pro-oncogenic protein absent from the majority of body tissues (with exception of brain, heart and skeletal muscle1), but expressed in many cancers1-3, where it provides tumor cells with improved fitness and survival. Though its canonical function is delivering aminoacyl-tRNAs to the ribosome, other “moonlighting” functions such as enhancing sphingosine kinase4,5 or antioxidant (most probably through peroxiredoxin-1 stimulation) activities6 have been described for the elongation factor. Recently, we have reported that eEF1A2 is the target for plitidepsin, a marine-derived cyclic depsipeptide currently under development for the treatment of relapsed or refractory multiple myeloma patients7. We have also confirmed that eEF1A2 interacted with previously described partners as PRDX1 and SPHK and enhanced their pro-survival activities8. Here we investigated the role of new “moonlighting functions” of eEF1A2 in the maintenance of the tumor phenotype and survival of cancer cells. Through co-immunoprecipitation and HPLC/MS we have uncovered the interaction between eEF1A2 and dsRNA-activated protein kinase (PKR, EIF2AK2). We have analyzed the kinase activity of PKR in the presence of eEF1A2, demonstrating that PKR activity is inhibited when complexed with eEF1A2. This complex is disrupted after plitidepsin binding to eEF1A2, rendering PKR active. Once activated, the kinase triggers a MAPK cascade and the NF-κB signaling pathway, leading to the activation of the extrinsic apoptotic pathway and the death of the tumor cell. Taken together, these results show that the fitness boost that the moonlighting functions of eEF1A2 provide to cancer cells, which are important for their growth and survival, constitutes an Achilles heel that can be purposely exploited in anticancer therapy. 1 Abbas, W., Kumar, A. & Herbein, G. Front Oncol 5, 75 (2015). 2 Anand, N. et al. Nature genetics 31, 301-305 (2002). 3 Li, Z. et al. PloS one 5, e10755 (2010). 4 Leclercq, T. M., Moretti, P. A., Vadas, M. A. & Pitson, S. M. J Biol Chem 283, 9606-9614 (2008). 5 Leclercq, T. M., Moretti, P. A. & Pitson, S. M. Oncogene 30, 372-378 (2011). 6 Chang, R. & Wang, E. J Cell Biochem 100, 267-278 (2007). 7 Losada, A. et al. Sci Rep 6, 35100 (2016) 8 Losada, A. et al. Abstract #1165, AACR Annual Meeting (2017) Citation Format: Juan F. Martínez-Leal, Alejandro Losada, Maria Jose Muñoz, Marta Martinez-Diez, Juan Manuel Dominguez, Carlos M. Galmarini. eEF1A2 interacts with and inhibits PKR to enhance cancer cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2906.

Introduction: Advanced GIST is commonly treated with tyrosine kinase inhibitors (TKI) [e.g. imatinib (IMA)]. With time the vast majority of patients develops TKI-resistance. GIST is generally believed to be resistant to chemotherapy with cytotoxic agents. The aim of our study was to test plocabulin (PLO; PM060184, PharmaMar), a potent cytotoxic tubulin-dynamics modifier, in two PDX models of GIST, characterized by different sensitivity to IMA. Experimental set-up: NMRI nu/nu mice (n=34) were transplanted bilaterally with human xenografts UZLX-GIST3sens (KIT: exon 11 p.W557_V559delinsF; IMA-sensitive) or -GIST9res (KIT: exon 11+17: p.P577del;W557LfsX5;D820G; IMA-resistant). Xenografted animals were randomly assigned to three treatment groups: control [vehicle, 5ml/kg/QW intravenously (i.v.)], IMA (50mg/kg/BID orally) and PLO (16mg/kg/QW, i.v.). Treatment lasted 22 days and the antitumor activity was assessed by tumor volume measurement, histopathology and KIT signaling pathway by Western blotting. Histological response (HR) was evaluated as previously described by Antonescu et al. 2005. Mann Whitney U test was used for statistical analysis with p Results: PLO treatment resulted in a reduction of tumor volume to 59% in GIST3sens and to 70% of the baseline volume in the GIST9res model. Good HR (grade 3, 4) was observed in 70% (GIST3sens) and 50% (GIST9res) of tumors. HR obtained with PLO was mainly characterized by necrosis, while IMA produced mainly myxoid degeneration in the sensitive model. In addition, in GIST3sens PLO decreased the microvessel area and increased apoptosis as assessed by immunohistochemistry, which was not observed in GIST9res. In the latter model PLO showed better activity than IMA in terms of tumor volume reduction (59% vs. 146%, p Conclusions: PLO is the first anti-tubulin agent showing antitumor activity in GIST PDX, both in models sensitive or resistant to IMA. The drug causes cytotoxicity in GIST, mainly through necrosis, without affecting KIT signaling. Due to the different modes of action of PLO and established TKI our work provides a scientific rationale to combine PLO and IMA to overcome resistance to small molecule TKI. Citation Format: Agnieszka Wozniak, Yannick Wang, Jasmien Wellens, Yemarshet K. Gebreyohannes, Maria Jose Guillén, Carlos M. Galmarini, Pablo M. Avilés, Maria Debiec-Rychter, Raf Sciot, Patrick Schöffski. Plocabulin, a tubulin inhibitor, presents antitumor activity in patient-derived xenograft (PDX) models of gastrointestinal stromal tumor (GIST) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3840.

Inflammatory mediators present in the tumor milieu may promote cancer progression and are considered promising targets of novel biological therapies. We previously reported that the marine antitumor agent trabectedin, approved in Europe in 2007 for soft tissue sarcomas and in 2009 for ovarian cancer, was able to downmodulate the production of selected cytokines/chemokines in immune cells. Patients with myxoid liposarcoma (MLS), a subtype characterized by the expression of the oncogenic transcript FUS-CHOP, are highly responsive to trabectedin. The drug had marked antiproliferative effects on MLS cell lines at low nanomolar concentrations. We tested the hypothesis that trabectedin could also affect the inflammatory mediators produced by cancer cells. Here, we show that MLS express several cytokines, chemokines, and growth factors (CCL2, CCL3, CCL5, CXCL8, CXCL12, MIF, VEGF, SPARC) and the inflammatory and matrix-binder protein pentraxin 3 (PTX3), which build up a prominent inflammatory environment. In vitro treatment with noncytotoxic concentrations of trabectedin selectively inhibited the production of CCL2, CXCL8, IL-6, VEGF, and PTX3 by MLS primary tumor cultures and/or cell lines. A xenograft mouse model of human MLS showed marked reduction of CCL2, CXCL8, CD68+ infiltrating macrophages, CD31+ tumor vessels, and partial decrease of PTX3 after trabectedin treatment. Similar findings were observed in a patient tumor sample excised after several cycles of therapy, indicating that the results observed in vitro might have in vivo relevance. In conclusion, trabectedin has dual effects in liposarcoma: in addition to direct growth inhibition, it affects the tumor microenvironment by reducing the production of key inflammatory mediators. Cancer Res; 70(6); 2235–44

BACKGROUND: Ewing sarcoma (ES) is a pediatric malignancy characterized by the EWS-FLI1 transcription factor. ES tumors require the continuous transcriptional activity of EWS-FLI1 for the oncogenic phenotype, regardless of fusion type, which makes EWS-FLI1 an ideal therapeutic target. To date, no EWS-FLI1 directed therapies have been developed. Interestingly, there have been reports of ES patients as exceptional responders in clinical trials, including a complete response in a refractory, metastatic patient that was administered the natural product trabectedin in a Phase I trial. We have since established trabectedin as an EWS-FLI1 inhibitor that acts through a re-localization mechanism at concentrations that are clinically achievable. Intriguingly, trabectedin failed in the Phase II setting after a change in schedule that lead to a decrease in reported Cmax. In this report, we unify the disparate clinical trial conclusions by examining the schedule-dependency of trabectedin in ES cells. METHODS: Here, we model a broad range of exposures to trabectedin and investigate the cell viability kinetics using live cell imaging analysis software and traditional MTS. In parallel, we present a novel assay to quantitatively measure the kinetics of drug clearance in vitro. In addition, we use qPCR and western blotting to examine EWS-FLI1 target gene expression changes in response to different exposures to trabectedin. Finally, we investigated ES cell lines containing variant EWS-FLI1 fusion types and identify an increased sensitivity of type III/IV ES cell lines to trabectedin treatment. We recapitulated these findings in vivo using ES xenograft models. RESULTS: Sensitivity of ES cells to trabectedin treatment is schedule-dependent. Here, we demonstrate that cells treated with trabectedin at equivalent exposures but different Cmax exhibit profound differences in cell viability. The differences in viability correlate with Cmax that are necessary to inhibit EWS-FLI1 activity in vitro. By measuring the kinetics of drug clearance, we show that the inhibitory effects on EWS-FLI1 are durable even after trabectedin has been washed out of the culture medium. In addition, we examine the sensitivity of variant EWS-FLI1 fusion proteins such as type III/IV fusions both in vitro and in vivo. CONCLUSIONS: In this report, we use clinical trial data to develop a more clinically relevant cell culture system for ES drug treatments. Importantly, we demonstrate that equivalent exposures do not lead to equivalent outcomes. We believe that this observation explains the discordant results of the Phase I and II clinical trials, and suggests that the Cmax achieved in serum dictates whether a patient will respond. In addition, we show that type III/IV EWS-FLI1 fusion proteins are particularly sensitive to the drug. Citation Format: Matt Harlow, Elissa Boguslawski, Anderson Peck, Zachary Madaj, Maria J. Navarro, Pablo Aviles, Carlos Galmarini, Patrick Grohar. Optimizing the trabectedin-mediated inhibition of EWS-FLI1 activity by schedule and fusion type [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 LB-047. doi:10.1158/1538-7445.AM2017-LB-047

Lurbinectedin (PM1183) is a new synthetic compound from the tetrahydroisoquinoline family, which has demonstrated a strong antiproliferative activity against a panel of human tumor models in preclinical assays and is currently being evaluated in phase III clinical trials in platinum-resistant ovarian cancer and small cell lung cancer. Lurbinectedin binds to DNA, inhibits trans-activated transcription, induces the degradation of elongating RNA Pol II and fools nucleotide excision repair to produce dsDNA breaks that need to be repaired mainly by homologous recombination (HR)1,2. Nearly 70% of patients diagnosed with ovarian cancer are in advanced stage, and the vast majority of them will eventually relapse after a primary cytoreductive surgery and several cycles of standard adjuvant chemotherapy including a platinum drug and a taxane. After a period of treatment with platinum drugs, patients will finally develop resistance, usually mediated by mechanisms such as drug detoxification or efflux and enhanced DNA repair. IRF-1 transcription factor expression has been shown to be up-regulated by cisplatin (CDDP) in ovarian cancer cells and might be limiting the response to the drug, likely by inhibiting cell proliferation3. Here we took advantage of the A2780/A2780cis human ovarian cancer cell lines, the second being a cisplatin resistant derivative, to investigate the role of IRF1 in the response of human ovarian cancer cells to cisplatin and lurbinectedin. A2780cis cells are, indeed, more resistant to cisplatin that their parental cell line but they do not differ in their resistance to lurbinectedin. Basal IRF-1 protein levels were actually higher in A2780cis cells than in their parental cell line, contributing to their resistance to cisplatin. Furthermore, cisplatin treatment induced the overexpression and nuclear localization of IRF-1 both, in A2780 and A2780cis cell lines. Contrarily, lurbinectedin did not induce the overexpression of IRF-1 neither in A2780 nor in A2780cis, explaining why this latter cell line is not resistant to the compound. Furthermore, lurbinectedin co-treatment with cisplatin reduced the expression of IRF-1 in A2780 and, more importantly, in A2780cis cells, explaining the synergism the combination has on these tumor cell lines. Thus, lurbinectedin not only did not activate the same mechanisms of resistance as cisplatin in ovarian cancer cells, but even reversed the resistance of these resistant cells to platinum drugs. 1 Santamaría Nuñez et al, 2016. Mol Cancer Ther 15(10):2399-2412 2 Romano et al, 2013. Int J Cancer. 2013 Nov;133(9):2024-33 3 Pavan et al, 2013. Eur J Cancer 49(4):964-973 Citation Format: Gema Santamaria Nuñez, Maria Jose Guillén, Juan F. Martínez-Leal, Pablo Avilés, Carlos M. Galmarini. Lurbinectedin reverses platinum dependent IRF1 overexpression and nuclear localization, partially responsible for resistance to platinum drugs in ovarian cancer [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 1211. doi:10.1158/1538-7445.AM2017-1211

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 Muñoz, Juan F. Martínez-Leal, Juan M. Domínguez, 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

Vascular supply of tumors is one of the main targets for cancer therapy. Different studies have shown that most of the microtubule-binding agents present antiangiogenic and vascular-disrupting effects. PM060184 is a new marine-derived drug that binds to a new site in β-tubulin inhibiting tubulin polymerization. The compound is currently being evaluated in phase 1/2 studies in patients with advanced malignancies. The present study describes the antiangiogenic and vascular-disrupting properties of PM060184. Treatment of HUVEC endothelial cells with PM060184 resulted in depolymerization of the microtubule network. Differently from untreated cells, PM060184-treated cells showed a rounded morphology with a well-developed actin filament structure and plasma membrane blebs. Using transwell chambers pre-coated with matrigel™ or collagen matrix, we showed that PM060184 inhibited migration and invasion of HUVEC endothelial cells. We finally found that PM060184 interfered with the correct formation of the HUVEC capillary network grown on matrigel™; moreover, drug treatment also resulted in a significant disruption of the previously formed capillary-like network. This rapid collapse of the endothelial tubular network in vitro occurred in a concentration-dependent manner and was observed at concentrations lower than those affecting cells survival. These in vitro findings were confirmed in nude mice bearing MDA-MB-231 (breast) xenografts: after the administration of a single dose of PM060184 (at its MTD, 16 mg/kg,) a very strong reduction in the number of vessels was quantified in serial tumor sections stained with H&E. Moreover, nude mice xenografted with H-460 (NSCLC) tumors and treated with a single dose of either 2 or 16 mg/kg of PM060184, experienced a strong and dose-dependent decrease of the intratumoral fluorescence after the administration of Angiosense™ 680, a fluorescence vascular imaging probe. Altogether, these data suggest that an anti-vascular mechanism is also contributing to the anti-tumor activities of PM060184. Citation Format: Carlos M. Galmarini, Maria J. Guillen, Juan F. Martinez-Leal, Marta Martinez-Diez, Pablo Aviles. Anti-angiogenic properties of PM060184. [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 3066.

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. Martínez-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.

BACKGROUND: Ewing sarcoma is a bone and soft tissue sarcoma characterized by fusion of the wild-type EWSR1 and FLI1 genes, which creates a constitutively activated transcription factor, EWS/FLI1. Ewing sarcoma cells are dependent on the continued activity of EWS/FLI1 to block differentiation and promote proliferation. For these reasons, EWS/FLI1 represents an ideal drug target. With this in mind, we have shown that the small molecule trabectedin inhibits EWS/FLI1 activity and synergizes with irinotecan in vivo. In addition, we showed that the gene expression response of Ewing sarcoma cells to trabectedin treatment is identical to keratinocytes exposed to UV light. Interestingly, wild-type EWSR1 accumulates in the nucleolus in response to UV light damage. Therefore, we hypothesize that the response and sensitivity of Ewing sarcoma cells to trabectedin is due to the re-localization of EWS/FLI1 in the nucleus. METHODS: Here, we utilize confocal microscopy and chromatin immunoprecipitation to characterize the localization of EWS/FLI1 and EWS/FLI1 deletion mutants in response to trabectedin. Because trabectedin has a narrow therapeutic index, we characterize the effect of PM01183, a second-generation structural analog with an improved pharmacokinetic profile, on EWS/FLI1 localization. In addition, we use qPCR and western blotting to determine the kinetics of EWS/FLI1 inhibition in response to this class of compounds. W demonstrate the effects of PM01183 treatment on the EWS/FLI1 gene signature genome-wide. Finally, we translate these findings in vivo using two Ewing sarcoma xenograft models. RESULTS: Treatment of Ewing sarcoma cells with Trabectedin and PM01183 causes EWS/FLI1 to accumulate in the nucleolus. This effect is accompanied by a loss of binding at target sequences and a reversal in expression of the gene signature of EWS/FLI1. We found the effect on EWS/FLI1 to be sustained, even after removal of the drug from the cell culture medium. We extended these findings in vivo to demonstrate the efficacy of a previously characterized combination therapy. Interestingly, treatment of Ewing sarcoma xenografts with the combination therapy caused the tumor to be replaced by benign adipocytes. CONCLUSIONS: We characterize a novel method of targeting an oncogenic fusion protein by leveraging a property of the wild-type protein to inactivate the oncogenic transcription factor. We demonstrate that wild-type EWSR1 protein's propensity to accumulate in the nucleolus upon UV light damage is maintained within the oncogenic EWS/FLI1 protein with trabectedin or PM01183 treatment. We believe that this accounts for the heightened sensitivity of Ewing sarcoma cells to this class of compounds and has numerous clinical implications. Citation Format: Matt Harlow, Matthew Easton, Maria Jose Guillén Navarro, Lisa Turner, Galen Hostetter, Carlos Galmarini, Pablo Aviles, Patrick Grohar. PM01183 inactivates the EWS/FLI1 transcription factor by redistributing the protein within the nucleus. [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 LB-177.

Cancer cells are characterized for their avid demand for active transcription, which reaches the level of a real addiction in solid tumors as small cell lung cancer (SCLC) or triple-negative breast cancer. Pharmacological modulation of transcription may thus provide a therapeutic approach to treat tumor types that depend on deregulated transcription for the maintenance of their oncogenic state. Lurbinectedin, currently under evaluation in a Phase III clinical trial for platinum-resistant ovarian cancer patients, and with very promising activity in combination with doxorubicin in SCLC, inhibits active transcription. Here we demonstrate that, after binding to specific DNA triplets highly represented in the CG-rich region surrounding the promoter of genes, this drug induces a rapid degradation of RNA Polymerase II (Pol II). Our results show that the hyperphosphorylated form of Pol II, already engaged in transcription elongation and likely blocked during this process by the lurbinectedin-DNA adduct, is then specifically submitted to the ubiquitin/proteasome degradation process, which finally removes the majority of the Pol II protein pool in treated cells. Disappearance of Pol II is followed by the formation of DNA breaks, process in which the nucleotide excision repair (NER) machinery, specifically the endonuclease XPF, has an important role. Pol II degradation and subsequent DNA damage were not only abrogated by inhibitors of CDK7 and CDK9 cyclin dependent kinases (DRB and flavopyridol), ubiquitin ligation (PYR-41), or proteasome activity (MG132), but also correlated with the antiproliferative activity of lurbinectedin in different cancer cell line models. In summary, lurbinectedin exemplifies a prototype drug for targeting transcriptional dependency in tumor cells and, thus, it could represent a new therapeutic alternative for solid tumors with this addiction. Citation Format: Gema Santamaria-Nunez, Carlos M. Genes-Robles, Juan F. Martínez-Leal, Carlos M. Galmarini, Jean Marc Egly. Lurbinectedin specifically targets transcription in cancer cells, triggering DNA breaks and degradation of phosphorylated Pol II. [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 3039.

Lurbinectedin, currently undergoing clinical evaluation in ovarian, breast and small-cell lung cancer patients, inhibits active transcription. The drug is structurally related to trabectedin containing the same pentacyclic skeleton of the fused tetrahydroisoquinoline rings, but differing by the presence of a tetrahydro-B-carboline replacing the additional tetrahydroisoquinoline of trabectedin. We investigated whether lurbinectedin has the ability to regulate the inflammatory tumor microenvironment in vitro and in vivo. Human purified monocytes were highly sensitive to lurbinectedin (IC50: 5-10 nM) and, after drug treatment, underwent caspase-8-dependent apoptosis. Furthermore, in vitro, lurbinectedin significantly inhibited the production of selected inflammatory chemokines (CCL2, CXCL8) and VEGF by stimulated monocytes and liposarcoma cell lines. Administration of lurbinectedin to mice bearing a murine fibrosarcoma -resistant to this compound in vitro- resulted in significant anti-tumor activity (T/C value around 50%). The analysis of immune cells of blood spleen and tumor tissues during treatment with lurbinectedin revealed a significant and selective decrease in the subset of monocytes and macrophages, including tumor-associated macrophages (TAM). A gene expression analysis of monocytes treated with lurbinectedin indicated a modulation of the transcriptional program in LPS-stimulated human monocytes. Overall, these results indicate that lurbinectedin affects the inflammatory micro-environment, with a selective apoptotic-inducing effect on mononuclear phagocytes and a specific inhibition of production of inflammatory cytokines. Citation Format: Paola Allavena, Cristina Belgiovine, Manuela Liguori, Ezia Bello, Roberta Frapolli, Carlos M. Galmarini, Maurizio D’Incalci. Lurbinectedin reduces tumor-associated macrophages and the production of inflammatory cytokines, chemokines, and angiogenic factors in preclinical models. [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 1284.

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 José Muñoz, Carolina Garcia, Juan F. Martínez-Leal, Federico Gago, Carmen Cuevas, Luis F. Garcia-Fernández, 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

Plitidepsin is a new marine-derived anti-tumor agent, originally isolated from the tunicate Aplidium albicans that is now fully obtained by total chemical synthesis. The compound is currently being evaluated in a pivotal phase III trial in patients with refractory/relapsed multiple myeloma. Previous in vitro studies in 26 hematological cell lines demonstrated that plitidepsin was more active in multiple myeloma and non-Hodgkin lymphoma compared to CML, AML and ALL derived cell lines as determined in a 4 day proliferation assay in suspension cultures. We have recently shown that plitidepsin interacts selectively with eEF1A2 (eukaryotic translation elongation factor 1 alpha 2) in tumor cells, which results in apoptotic cell death. To identify target tumor types for future clinical studies, the inhibition of colony formation of plitidepsin was assessed in vitro in 116 established patient derived xenografts tumor models from 23 different tumor types, including solid tumors and hematological malignancies such as myeloma, leukemia and lymphoma. The anti-tumor activity of plitidepsin varied depending on the concentration used, achieving a mean IC70 (inhibitory concentration of 70%) value of 3 nM (range between 0.03 nM and >1 μM), which can be also achieved in patients treated using the clinical recommended dose or less. 39 out of 116 tumors showed above average sensitivity (IC70 < 1nM) and IC70 values in these tumors were about 9-fold lower than the mean of all tumors as tested. Sensitive tumor types were seen among different tumor types, such as hematological malignancies, pleuramesothelioma, melanoma, small cell and non-small cell lung cancer (both adeno and large cell), as well as renal cancer. In conclusion plitidepsin showed a broad differential antitumor activity in vitro in hematological malignancies and in solid tumors. Bioinformatic analyses are currently underway to identify biomarkers and/or gene signatures that can predict sensitivity or resistance to plitidepsin for selecting patients being responsive to the drug. Citation Format: Armin Maier, Gerhard Kelter, Vincent Vuaroquaux, Pablo M. Avilés Marin, Carmen Cuevas, Carlos M. Galmarini, Heinz H. Fiebig. Plitidepsin shows antitumor activity in patient-derived tumor xenografts and hematologic malignancies. [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 4472. doi:10.1158/1538-7445.AM2015-4472

Background: Antibody-drug conjugates (ADC`s) have emerged as powerful tools for the treatment of cancer, combining the potency of cytotoxic molecules with the selectivity of antibodies targeted towards specific antigens. Marine compounds represent an interesting opportunity worth exploring as they possess the requirements needed to be considered promising payloads. The in vivo results obtained with a new ADC, MI130004, in HER2 expressing breast tumors are presented here. Materials and Methods: To evaluate the long lasting antitumor effect induced by MI130004, immunodefficient female mice were subcutaneously implanted with HER2 expressing breast lines namely, JIMT-1, BT-474 and MAXF574.Tumor (ca. 100 mm3) bearing animals were randomly allocated (Day 0) to receive MI130004 (10mg/kg), trastuzumab (30 or 10 mg/kg) or placebo (N = 8-10/group). Intravenous treatments were weekly administered for 5 consecutive weeks and then, tumor volume growth was recorded 2-3 times per week. For survival evaluation, time to endpoint was define as the time from Day 0 to death as a results of tumor growth (>2000 mm3) or any other cause (e.g., tumor necrosis). Statistical differences were assessed by Kaplan-Meier curves with the log rank test. The follow-up period was extended until 90 (MAXF574) or 120 days (JIMT-1 and BT-474) after the initiation day (Day 0). Then, surviving animals were sacrificed, tumor (or skin around the former tumor site) dissected free, formalin fixed and paraffin embedded for histopathology evaluations. Results: The treatment with MI130004 induced a long lasting antitumor effect with statistically significant increases in median survival time compared to either placebo (P = 0.0336, P = 0.0001 and P Conclusions: The treatment with MI130004 on mice bearing HER2 expressing breast tumors resulted in strong, long lasting antitumor activity, including complete tumor remissions. These results strongly suggest that MI130004 is a new ADC with extraordinary therapeutic anti-cancer properties. Citation Format: Pablo M. Aviles, Maria Jose J. Guillen, Alberto Gallardo, Maria Virtudes Cespedes, Ramon Mangues, Heiner Fiebig, Natalie Hartman, Juan Manuel Dominguez, Luis Francisco Garcia, Carlos Galmarini, Carmen Cuevas. MI130004, a new antibody-drug conjugate, induces strong, long-lasting antitumor effect in HER2 expressing breast tumor models. [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 2480. doi:10.1158/1538-7445.AM2015-2480

Lurbinectedin (PM1183) is a new tetrahydroisoquinoline derivative currently evaluated in phase II trials in BRCA1-mutated breast cancer and NSCLC patients. Two pivotal trials (in platinum-resistant ovarian cancer and in SCLC) will be launched in 2015. PM1183 shows antitumor activity against a wide variety of tumor cells with mean IC50 values in the low nanomolar range. In living cells, PM1183 inhibits trans-activated transcription and blocks NER-dependent DNA repair inducing the formation of dsDNA breaks and the collapse of replication forks that need to be repaired by homologous recombination (HR). As expected, PM1183 is more active against homologous recombination (HR)-deficient cell lines. In this work, we evaluated the in vitro antitumor effect of PM1183 when combined with the PARP inhibitors ABT-88 (Veliparib, PARP-1 and -2), AZD-2281 (Olaparib, PARP-1), BSI-201 (Iniparib, PARP-1) and BMN-673 (PARP-1 and -2) (Selleck Chemicals, Houston, TX, USA) in a panel of human breast carcinoma cells with different BRCA1 status, including MCF-7 (BRCA1 +/+), MDA-MB-231 (BRCA1 +/-), HCC-1937 (BRCA1 -/-) and MDA-MB-436 (BRCA1 -/-). The combination index (CI) of each different drug-drug combination was calculated by applying the Chou and Talalay method. Different degrees of synergism (CI < 1) were recorded when PM1183 was combined with AZD-2281 (Olaparib) and BMN-673, depending on the tumor cell line and the conditions tested. The highest degree of synergism with Olaparib was observed in MDA-MB-436 cells followed by MDA-MB-231, MCF7 and HCC-1937 cells. The highest degree of synergism with BMN-673 was observed in MCF-7 cells followed by MDA-MB-231 and HCC-1937 cells. In MDA-MB-231 cells, both combinations led to a synergistic induction of γ-H2AX expression and PARP-1 cleavage, as analyzed by western blot. Collectively, our results indicate that the combination of PM1183 and PARP inhibitors (Olaparib and BMN673) induces an artificial synthetic lethality that can be advantageously used to kill several breast cancer cells, independently from their BRCA1 status. Citation Format: Gema Santamaría, Sonia Avila, Victoria Moneo, Carmen Cuevas, Luis F. García-Fernández, Carlos M. Galmarini. Synergistic combination of lurbinectedin and PARP inhibitors in breast cancer tumor cell lines. [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 2520. doi:10.1158/1538-7445.AM2015-2520

BACKGROUND: Ewing sarcoma is a pediatric malignancy characterized by the fusion of the EWSR1 and FLI1 genes, which creates a constitutively activated transcription factor, EWS/FLI1. It is widely known that Ewing sarcoma cells depend on the transcriptional program of EWS/FLI1 for cell survival. Therefore, the goal of this research is to develop and clinically translate small molecules that suppress EWS/FLI1 activity. We have previously reported that the natural product trabectedin suppresses EWS/FLI1 activity. In addition, the compound synergizes with irinotecan to suppress Ewing sarcoma tumor growth. However, the compound suffers from a narrow therapeutic index that limits the poisoning of EWS/FLI1 in patients. In this report, we show that the trabectedin analog PM01183 has improved targeting of EWS/FLI1 and preserved synergy with irinotecan. In addition, the compound is known to have a dramatically improved therapeutic index suggesting improved activity of this compound in the clinic. METHODS: In this report, we characterize the mechanism of suppression of EWS/FLI1 by trabectedin using confocal immunocytochemistry and chromatin immunoprecipitation. We show enhanced suppression of EWS/FLI1 by PM01183 using the Fluidigm platform and confirm the results with a luciferase reporter construct and high-content quantitative PCR. Finally, we tested the ability of PM01183 to suppress tumor growth in xenograft models of Ewing sarcoma both in the absence and presence of irinotecan. RESULTS: PM01183 more effectively suppressed the gene signature of EWS/FLI1 than the parent compound trabectedin. In addition, the drug re-localizes EWS/FLI1 away from target genes in the nucleus leading to suppression of these EWS/FLI1 targets. These results are consistent with the activity of the parent compound, trabectedin, that blocks binding of EWS/FLI1 to chromatin. More importantly, the drug causes a regression of a TC32 xenograft and markedly suppresses growth of a TC71 xenograft when combined with irinotecan. Finally, PM01183 has a substantially improved pharmacologic profile in patients in comparison to trabectedin suggesting that these effects are bio-achievable in patients. CONCLUSIONS: In comparison to trabectedin, we have shown that its structural analog, PM01183 shows enhanced suppression of an EWS/FLI1 gene signature and preserves the synergy with irinotecan that we previously reported. We suggest a mechanistic basis for this activity against EWS/FLI1 and show excellent activity against Ewing sarcoma xenografts. Together, these results suggest that the clinical translation of PM01183 as an EWS/FLI1 targeted therapy both alone and in combination with irinotecan is warranted. Citation Format: Matt Harlow, Nichole Maloney, Maria Jose Guillen Navarro, Maurizio D'Incalci, Carlos Galmarini, Pablo Manuel Aviles Marin, Patrick Grohar. PM01183 shows an improved therapeutic index relative to trabectedin and suppresses EWS/FLI1 activity at clinically achievable concentrations. [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 3962. doi:10.1158/1538-7445.AM2014-3962

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

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

Background: Lurbinectedin (PM01183), a new synthetic tetrahydroisoquinoline alkaloid, is a minor groove specific DNA binder that interacts directly with specific factors involved in DNA repair and transcription pathways. In living cells, PM01183-DNA adducts stall replication and transcription giving rise to double strand breaks, inducing accumulation of cells in the S-phase of the cell cycle and triggering apoptosis. In both the ongoing phase II trials (pancreas and platinum-resistant ovarian cancer) the first stage was concluded with positive results in clinical activity, ensuring the continuity of the trials. Also, phase I trials in combination with doxorubicin and gemcitabine as well as in advanced acute leukemia are ongoing. The in vitro and in vivo synergism of PM01183+ topoisomerase I inhibitors is presented here. Material and Methods: In vitro: PM01183+topotecan synergism was tested in a panel of 12 tumor cell lines. In vivo: Athymic mice were implanted with SW1990 (pancreas), HT29 (colon) or H460 (NSCLC) cancer cells. Mice bearing HT29 or H460 tumors (ca. 150 mm3) were allocated to 13 groups (N=6-8/group): placebo; PM01183 at MTD (0.180 mg/kg [0.54 mg/m2]), 0.75MTD, 0.5MTD and 0.25MTD; irinotecan at MTD (50 mg/kg [150 mg/m2]), 0.75MTD, 0.5MTD and 0.25MTD; and, PM01183+irinotecan at (1+1), (0.75+0.75), (0.50+0.50) and (0.25+0.25) MTDs. Animals bearing SW1990 tumors received treatments (single or combination) at the MTD levels. Treatments were given intravenously once per week (PM01183) or every 4 days (irinotecan) during the placebo-treated survival time. The combination index (CI) was determined by the CI-isobol method. Results: The combination of PM01183 plus topotecan in NSCLC (A549), melanoma (SK-MEL-2), hepatocarcinoma (HepG2), colon (HT29), pancreas (PANC-1) and glioma (U87MG) cells was considered synergistic. In vivo, a synergistic antitumor effect (CI Conclusion: PM01183 synergizes the in vitro effect of topotecan in several tumor cell lines including NSCLC, melanoma, hepatocarcinoma, colon, pancreas and glioma. In agreement with the in vitro results, the treatment of mice xenografted with colon, NSCLC or pancreas tumors with PM01183 combined with a topoisomerase I inhibitor (irinotecan) resulted in synergistic antitumor activity. Citation Format: Carlos Galmarini, Maria José Guillen, Victoria Moneo, Luis Francisco Garcia, Mandy Palomares, Oscar Cataluña, Carmen Cuevas, Pablo Avilés. Lurbinectedin (PM01183) synergizes with topoisomerase I inhibitors in vitro and in vivo. [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 5499. doi:10.1158/1538-7445.AM2013-5499

Lurbinectedin (PM01183), a new synthetic tetrahydroisoquinoline alkaloid, is a minor groove specific DNA binder that interacts directly with specific factors involved in DNA repair and transcription pathways. In living cells, PM01183-DNA adducts stall replication and transcription giving rise to double strand breaks, inducing accumulation of cells in the S-phase of the cell cycle and triggering apoptosis. In both the ongoing phase II trials (pancreas and platinum-resistant ovarian cancer) the first stage was concluded with positive results in clinical activity, ensuring the continuity of the studies. Also, phase I trials in combination with doxorubicin and gemcitabine as well as in advanced acute leukemia are ongoing. Here, we examined the effects of PM01183 on the activity and stability of the RNAPol II as well as on other factors of the transcriptional machinery, including TBP (TFIID), p62 (TFIIH), XPG and XPF. Our results showed that PM01183 induced a rapid, time- and concentration-dependent degradation of RNA Pol II in a panel of different human tumor cell lines, including HCT-116 (colon), A549 (NSCLC), HeLa (cervix) and A763 (sarcoma). This degradative process was efficiently abrogated in the presence of transcriptional (DRB or Actinomycin D) or proteasome inhibitors (MG132), demonstrating that PM01183 specifically targets the transcriptionally active RNA Pol II for degradation via the proteasome pathway. In addition, it was also shown that the effect of PM01183 on the RNA Pol II was dependent on the presence of a functional TC NER repair machinery. PM01183 induced degradation of RNA Pol II in global NER (XPC) deficient cells, but failed to do it in TC NER (CSB, XPD and XPG) deficient cells. Importantly, these effects were confirmed to be specific for the RNA Pol II, since other factors of the transcriptional machinery, such as TBP (TFIID), p62 (TFIIH), XPG or XPF or the RPA194 subunit of the RNA Pol I were not affected. Together, these results show up the complex mechanism of action of the drug on tumor cells. Citation Format: Gema Santamaria, Juan F. Martinez-Leal, Carmen Cuevas, Luis F. Garcia-Fernandez, Carlos M. Galmarini. Lurbinectedin (PM01183) specifically targets RNA Pol II for degradation via the proteasome pathway in a TC-NER-dependent fashion. [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 5558. doi:10.1158/1538-7445.AM2013-5558

Background: Lurbinectedin (PM01183), a new synthetic tetrahydroisoquinoline alkaloid, is a minor groove specific DNA binder that interacts directly with specific factors involved in DNA repair and transcription pathways. In living cells, PM01183-DNA adducts stall replication and transcription giving rise to double strand breaks, inducing accumulation of cells in the S-phase of the cell cycle and triggering apoptosis. In both the ongoing phase II trials (pancreas and platinum-resistant ovarian cancer) the first stage was concluded with positive results in clinical activity, ensuring the continuity of the trials. Also, phase I trials in combination with doxorubicin and gemcitabine as well as in advanced acute leukemia are ongoing. The synergism (in vitro and in vivo) of PM01183 combined with 5-Fluorouracil (5-FU) is presented here. Material and Methods: In vitro: The combination of PM01183+5-FU was tested in 12 different tumor cell lines: lung, sarcoma, melanoma, prostate, pancreas, gastric, ovarian, hepatocarcinoma, colon, kidney, glioma and breast cancer. In vivo: Athymic mice were implanted with HGC-27 (gastric) cancer cells. Mice bearing tumors (ca. 150 mm3) were allocated to 13 groups (N=6-8/group): placebo; PM01183 at MTD (0.180 mg/kg [0.54 mg/m2]), 0.75MTD, 0.5MTD and 0.25MTD; 5-FU at MTD (50.0 mg/kg), 0.75MTD, 0.5MTD and 0.25MTD; and, PM01183+5-FU at (1+1), (0.75+0.75), (0.50+0.50) and (0.25+0.25) MTDs. In separate experiments, mice bearing SW1990 (pancreas) or HT-29 (colon) tumors received treatments (single or combination) at the MTD levels. All treatments were given intravenously once per week during the placebo-treated survival time. The combination index (CI) was determined by the CI-isobol method in the HGC-27 experiments (in vitro or in vivo). Statistical differences for the antitumor effect recorded between groups were determined using two-tailed Mann-Whitney U test. Results: In vitro, a synergistic effect was seen in gastric (HGC-27) and colon (HT29) cells. The recorded in vivo antitumor effect indicated synergism (CI Conclusion: In vitro, the combination of PM01183+5-FU showed a synergistic effect in gastric and colon tumor cell lines. In agreement, in vivo synergism was seen after the treatment with PM01183+5-FU of mice bearing gastric, colon or pancreas xenografted tumors. Citation Format: Pablo Aviles, Maria José Guillen, Carlos Galmarini, Luis Francisco García, Raquel López, Práxedes Núñez, Victoria Moneo, Carmen Cuevas. Synergism of lurbinectedin (PM01183) combined with 5-fluorouracil (5-FU): in vitro and in vivo studies. [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 5498. doi:10.1158/1538-7445.AM2013-5498

Irvalec® is a marine derived synthetic depsipeptide showing cytotoxic activity against a wide diversity of tumor cells both in vitro and in vivo. The compound is currently undergoing Phase II clinical studies in patients with solid tumors. The existing data on the mechanism of action of Irvalec® indicate that the compound inserts and self-organize in the plasma membrane of tumor cells, rapidly inducing loss of membrane integrity and cell permeabilization, giving rise to a fast and dramatic necrotic death. By continuous exposure of human colon carcinoma HCT-116 cells to stepwise increasing concentratrions of Irvalec®, a resistant subclone of the cell line was established, named HCT-116-Irv. In dose-response cytotoxicity assays, HCT-116-Irv cells showed a GI50 value >50 μM, representing a specific resistance index to the compound greater than 10.8 as compared to their wt counterpart. The resistant cells did not show any of the morphological and physiological responses to Irvalec® observed in parental cells and no overexpression of Pgp efflux pump or significant cross-resistance to other antitumoral drugs was detected. Due to the presumably direct effect of Irvalec® on the cell membrane, we searched for possible changes in the lipid composition of the resistant cells, acquired during the process of resistance development. After fractionation of the lipid extracts using thin layer cromatography (TLC), two differential fractions, that were completely absent in the resistant cells as compared to wt cells, were identified and purified. Interestingly, one of these fractions, named F4, was found to be able to interact with a biotinilated derivative of Irvalec®, using a standard overlay assay. These results suggested that the lipid composition could be directly related to the sensitivity of tumor cells to Irvalec®. Efforts are now focused on the chemical characterization of the selected lipid fraction and its putative biological role mediating the cytotoxic response to Irvalec®. 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 3805. doi:1538-7445.AM2012-3805

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

Irvalec®, a marine-derived depsipeptide, has showed potent cytotoxic activity in vitro against a wide variety of human tumors, including breast, colon, pancreas, lung and prostate. Irvalec® is currently undergoing phase II clinical studies in non-small lung cancer. Exposure of tumor cells to Irvalec® induces necrotic cell death, a process associated with rapid loss of membrane integrity and subsequent cell permeabilization. In dose-response experiments using different tumor cell lines, very similar IC50 values were obtained after short (30 min) and long (72 h) exposure times to the drug, suggesting that the compound exerts its cytotoxic effect immediately after drug treatment. Tumor cells exposed to effective concentrations of Irvalec® underwent rapid and dramatic morphological changes, including cell blebbing, severe swelling, and plasma membrane permeabilization (as detected by propidium iodide (PI) uptake and LDH release) that irrevocably lead to cell lysis. Interestingly, membrane damage was observed only after reaching a threshold concentration of around 1-5 µM and only in the continuous presence of the compound in the culture medium. Compounds acting at the cell membrane have been reported to have similar cellular effects than those observed with Irvalec®. The activity of these compounds was inhibited by the use of different cell membrane protectors. To test if these protectors exert some effect against the cytotoxic activity of Irvalec® we exposed A549 cells to a panel of 20 different compounds and then treated with Irvalec®. The cytotoxic activity of the drug was evaluated by PI nuclear staining by fluorescence microscopy, time-lapse videomicroscopy and plate fluorimetry. Among the compounds tested, only DIDS (dihydro-4,4′ diisothiocyanostilbene-2,2′-disulphonic acid) and zinc salts resulted in a drastic reduction of the early effects of Irvalec® in A549 cells. In the presence of DIDS or zinc, a 70% and 90% decrease was observed, respectively, in the cytotoxicity of the drug. Altogether, these results suggest that Irvalec® induces a rapid hydroelectrolitic disbalance in tumor cells, causing severe alterations that lead to a necrotic cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4454.

Irvalec® is a marine-derived antitumor agent that is currently undergoing phase II clinical trials. The compound induces a necrotic cell death associated with the appearance of membrane blebs and severe cell swelling reflecting a serious membrane damage caused by the drug. Indirect evidence has also suggested a possible role of ErbB proteins, in particular ErbB3, and lipid rafts in conferring sensitivity to Irvalec treatment. In order to assess the role of ErbB protein expression in the mechanism of action of Irvalec, and to measure the effect of drug treatment on the clustering properties of these proteins, we generated CHO cell lines stably transfected with ErbB2 (CHO-ErbB2) or with ErbB2 and ErbB3 (CHO-ErbB2-3). The IC50 values of the three cell lines for Irvalec did not differ significantly from each other. On the other hand treatment with Irvalec induced a significant increase in the binding of a conformational sensitive antibody to ErbB3 (ErbB3.105.5) without modifying the binding of other ErbB3 antibodies or antibodies against ErbB2. In addition, Irvalec treatment decreased the homoassociation of ErbB2 and ErbB3 measured by flow cytometric FRET. A431 cells transfected with GPI-anchored GFP or ErbB3-citrine showed significant Irvalec-induced patching of the GPI-anchor and ErbB3, whereas the distribution of ErbB1 and ErbB2 were not affected in cells transfected with ErbB1-GFP or ErbB2-mYFP. In conclusion, our data do not support a role for ErbB proteins in determining sensitivity to Irvalec, but show that these receptors are indirectly affected by exposing cells to the compound. These alterations should be ascribed to cell membrane modifications induced by Irvalec treatment Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4464.

Trabectedin is a marine derived antitumoral agent, originally isolated from Ecteinascidia turbinata. It acts by binding to the minor groove of DNA interfering with cell division, transcription, and the DNA repair machinery. Trabectedin was approved by the EMEA as second line therapy for the treatment of advanced soft tissue sarcoma and for ovarian cancer in combination with Doxil. Further Phase II trials with Trabectedin in breast and prostate cancer are underway. We characterized Trabectedin for antitumor efficacy and selectivity in patient-derived tumor xenografts to identify target tumor types for further clinical studies. The compound was tested in 67 tumor xenografts of 15 histo types using an ex vivo clonogenic assay. Pronounced concentration dependent antitumor activity (mean IC70 = 1.3 nM) and selectivity was observed, with sensitive tumor models being on average about 7-fold more sensitive than the average of all tumors tested. Trabectedin was also given to tumor-bearing nude mice at 0.2 and 0.15 mg/kg/d iv once weekly for 3 weeks and showed substantial inhibition of tumor growth at a dose level of 0.2 mg/kg/d in tumors of lung, colon, and breast. The activity data of Trabectedin against 67 tumors in the clonogenic assay were used for further bioinformatic analysis. Subsets of tumors and their corresponding data were randomly split into a training set (n=44) and an independent validation set (n=23). By matching in vitro antitumor efficacy data (IC70) of the tumors with the corresponding gene expression profiles (determined by Affymetrix HG-U133 Plus 2.0 gene chip array), a signature of 19 gene transcripts being specific for the responsiveness towards Trabectedin was determined. The classification border for activity of Trabectedin was IC70 = 0.5 nM. The signature was validated by leave-one-out-cross-validation (LOOCV) on the training set, and prediction of tumors of the independent validation set. In the LOOCV sensitivity or resistance of tumors was predicted correctly in 13/18 (72%) and 26/26 tumors (100%), in the validation set in 4/7 (57%) and 13/16 tumors (81%), respectively. In the validation set, the predicted responders showed a 4.5-fold lower median IC70 compared to the predicted non-responders (p= 0.02). Moreover, the signature was used to predict responsiveness of 173 tumors of the Oncotest xenograft collection with unknown sensitivity to Trabectedin. In this set of tumors, the signature identified sarcoma, leukemia/lymphoma, as well as ovarian, head and neck, small cell lung, mammary and bladder cancer as Trabectedin sensitive tumor types. Experimental testing of the predicted tumors so far confirmed the predictions in 11/13 cases (85%). This study shows the feasibility of combining experimental testing and virtual prediction to identify additional tumor types as candidates for further preclinical and clinical investigations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2598.