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Despite recent progress in its treatment, Multiple Myeloma (MM) remains incurable and its associated bone disease persists even after complete remission. Thus, identification of new therapeutic agents that simultaneously suppress MM growth and protect bone is an unmet need. Herein, we examined the effects of Aplidin, a novel anti-cancer marine-derived compound, on MM and bone cells. In vitro, Aplidin potently inhibited MM cell growth and induced apoptosis, effects that were enhanced by dexamethasone (Dex) and bortezomib (Btz). Aplidin modestly reduced osteocyte/osteoblast viability and decreased osteoblast mineralization, effects that were enhanced by Dex and partially prevented by Btz. Further, Aplidin markedly decreased osteoclast precursor numbers and differentiation, and reduced mature osteoclast number and resorption activity. Moreover, Aplidin reduced Dex-induced osteoclast differentiation and further decreased osteoclast number when combined with Btz. Lastly, Aplidin alone, or suboptimal doses of Aplidin combined with Dex or Btz, decreased tumor growth and bone resorption in ex vivo bone organ cultures that reproduce the 3D-organization and the cellular diversity of the MM/bone marrow niche. These results demonstrate that Aplidin has potent anti-myeloma and anti-resorptive properties, and enhances proteasome inhibitors blockade of MM growth and bone destruction.

Purpose This multicenter phase II trial evaluated lurbinectedin (PM01183), a selective inhibitor of active transcription of protein-coding genes, in patients with metastatic breast cancer. A unicenter translational substudy assessed potential mechanisms of lurbinectedin resistance. Patients and Methods Two arms were evaluated according to germline BRCA1/2 status: BRCA1/2 mutated (arm A; n = 54) and unselected ( BRCA1/2 wild-type or unknown status; arm B; n = 35). Lurbinectedin starting dose was a 7-mg flat dose and later, 3.5 mg/m2 in arm A. The primary end point was objective response rate (ORR) per Response Evaluation Criteria in Solid Tumors (RECIST). The translational substudy of resistance mechanisms included exome sequencing (n = 13) and in vivo experiments with patient-derived xenografts (n = 11) from BRCA1/2-mutated tumors. Results ORR was 41% (95% CI, 28% to 55%) in arm A and 9% (95% CI, 2% to 24%) in arm B. In arm A, median progression-free survival was 4.6 months (95% CI, 3.0 to 6.0 months), and median overall survival was 20.0 months (95% CI, 11.8 to 26.6 months). Patients with BRCA2 mutations showed an ORR of 61%, median progression-free survival of 5.9 months, and median overall survival of 26.6 months. The safety profile improved with lurbinectedin dose adjustment to body surface area. The most common nonhematologic adverse events seen at 3.5 mg/m2 were nausea (74%; grade 3, 5%) and fatigue (74%; grade 3, 21%). Neutropenia was the most common severe hematologic adverse event (grade 3, 47%; grade 4, 10%). Exome sequencing showed mutations in genes related to the nucleotide excision repair pathway in four of seven tumors at primary or acquired resistance and in one patient with short-term stable disease. In vivo, sensitivity to cisplatin and lurbinectedin was evidenced in lurbinectedin-resistant (one of two) and cisplatin-resistant (two of three) patient-derived xenografts. Conclusion Lurbinectedin showed noteworthy activity in patients with BRCA1/2 mutations. Response and survival was notable in those with BRCA2 mutations. Additional clinical development in this subset of patients with metastatic breast cancer is warranted.

In the search for novel payloads to design new antibody–drug conjugates (ADC), marine compounds represent an interesting opportunity given their unique chemical features. PM050489 is a marine compound that binds β-tubulin at a new site and disrupts the microtubule network, hence leading to mitotic aberrations and cell death. PM050489 has been conjugated to trastuzumab via Cys residues through a noncleavable linker, and the resulting ADC, named MI130004, has been studied. Analysis of MI130004 delivered data consistent with the presence of two molecules of PM050489 per antibody molecule, likely bound to both sides of the intermolecular disulfide bond connecting the antibody light and heavy chains. The antitumor activity of MI130004 was analyzed in vitro and in vivo in different cell lines of diverse tumor origin (breast, ovary, and gastric cancer) expressing different levels of HER2. MI130004 showed very high in vitro potency and good selectivity for tumor cells that overexpressed HER2. At the cellular level, MI130004 impaired tubulin polymerization, causing disorganization and disintegration of the microtubule network, which ultimately led to mitotic failure, mirroring the effect of its payload. Treatment with MI130004 in mice carrying histologically diverse tumors expressing HER2 induced a long-lasting antitumor effect with statistically significant inhibition of tumor growth coupled with increases in median survival time compared with vehicle or trastuzumab. These results strongly suggest that MI130004 is endowed with remarkable anticancer activity and confirm the extraordinary potential of marine compounds for the design of new ADCs. Mol Cancer Ther; 17(4); 786–94. ©2018 AACR.

Background: Lurbinectedin is a novel anticancer agent currently undergoing late-stage (Phase II /III) clinical evaluation in platinum-resistant ovarian, BRCA1/2-mutated breast and small-cell lung cancer. Lurbinectedin is structurally related to trabectedin and it inhibits active transcription and the DNA repair machinery in tumour cells. Methods: In this study we investigated whether lurbinectedin has the ability to modulate the inflammatory microenvironment and the viability of myeloid cells in tumour-bearing mice. Results: Administration of lurbinectedin significantly and selectively decreased the number of circulating monocytes and, in tumour tissues, that of macrophages and vessels. Similar findings were observed when a lurbinectedin-resistant tumour variant was used, indicating a direct effect of lurbinectedin on the tumour microenviroment. In vitro, lurbinectedin induced caspase-8-dependent apoptosis of human purified monocytes, whereas at low doses it significantly inhibited the production of inflammatory/growth factors (CCL2, CXCL8 and VEGF) and dramatically impaired monocyte adhesion and migration ability. These findings were supported by the strong inhibition of genes of the Rho-GTPase family in lurbinectedin-treated monocytes. Conclusions: The results illustrate that lurbinectedin affects at multiple levels the inflammatory microenvironment by acting on the viability and functional activity of mononuclear phagocytes. These peculiar effects, combined with its intrinsic activity against cancer cells, make lurbinectedin a compound of particular interest in oncology.

R-loops are a major source of replication stress, DNA damage, and genome instability, which are major hallmarks of cancer cells. Accordingly, growing evidence suggests that R-loops may also be related to cancer. Here we show that R-loops play an important role in the cellular response to trabectedin (ET743), an anticancer drug from marine origin and its derivative lurbinectedin (PM01183). Trabectedin and lurbinectedin induced RNA–DNA hybrid-dependent DNA damage in HeLa cells, causing replication impairment and genome instability. We also show that high levels of R-loops increase cell sensitivity to trabectedin. In addition, trabectedin led to transcription-dependent FANCD2 foci accumulation, which was suppressed by RNase H1 overexpression. In yeast, trabectedin and lurbinectedin increased the presence of Rad52 foci, a marker of DNA damage, in an R-loop–dependent manner. In addition to providing new insights into the mechanisms of action of these drugs, our study reveals that R-loops could be targeted by anticancer agents. Given the increasing evidence that R-loops occur all over the genome, the ability of lurbinectedin and trabectedin to act on them may contribute to enhance their efficacy, opening the possibility that R-loops might be a feature shared by specific cancers. Implications: The data presented in this study provide the new concept that R-loops are important cellular factors that contribute to trabectedin and lurbinectedin anticancer activity., This work was supported with funding by the European Research Council (ERC2014 AdG669898 TARLOOP), the Spanish Ministry of Economy and Competitiveness (BFU2016-75058-P), the European Union (FEDER; BFU2016-75058-P), and PharmaMar (PRJ201402213).

1548 Background: Synthetic fingerprints integrate clinical data within computational models allowing the identification of particular clinical subpopulations at a given moment. We here describe a deep learning strategy to detect super-responder and super-survivor patients with squamous NSCLC by setting up synthetic fingerprints and using unsupervised deep learning frameworks (UDLF). Methods: Through www.projectdatasphere.org, we accessed the control arm clinical data (N = 548) of the randomised phase III SQUIRE trial (NCT00981058). This trial included patients with stage IV squamous NSCLC who had not received previous chemotherapy. These patients were treated with gemcitabine 1,250 mg/m2 (IV, 30-min infusion, d1/d8) and cisplatin 75 mg/m2 (IV, 120 min infusion, d1) on a 3-week cycle for a maximum of six cycles. Synthetic fingerprints resulted of the integration of 180 features collected during the first 3 cycles including demographics, medical history, physical exam, concomitant medication, histopathology, PK parameters, adverse events and common labs. These fingerprints were used as input for the UDLF. The resultant clusters were correlated with overall-response rate (ORR) and overall survival (OS). Results: After missing data removal and feature standardization, 192 patients were eligible for the study. The UDLF was able to generate two different clusters: P0 (n = 107) and P1 (n = 84). ORR was higher in the P1 than in the P0 cluster (mean 41.6% [95% CI 31.7-52.3] vs. 28.0% [95% CI 20.4-37.2]; p = 0.04). OS was significantly longer in the P1 than in the P0 cluster (median 13.2 months vs. 9.7 months; hazard ratio 1.56 [95% CI 1.12-2.17; p = 0.008]). Feature contribution analysis showed that P1 had more patients and more events of grade III/IV neutropenia. In contrast, P0 had more patients and more events of grade III/IV nausea and vomiting. Other major differences were observed on vital signs (SBP, DBP, HR, RR, Temp), concomitant medication (osmotically-active laxatives, dexamethasone, furosemide, granisetron and ondansetron) and in hematological (RBC, HGB, HCT, MCV, WBC, neutrophils, monocytes, lymphocytes) and biochemistry (albumin, globulins, ALP, LDH, creatinine, BUN, urea, sodium, magnesium and phosphate) tests. Conclusions: Our findings show that synthetic fingerprints and subsequent deep learning analysis can be of use to identify patients with clinical characteristics associated with high-response rate and long-term survival.

1549 Background: Predicting the clinical course of metastatic disease remains a key challenge in CRC. Estimating prognosis of these late-stage patients can avoid undertreatment or overtreatment and also guide the follow-up intensity. This study has investigated the ability of an artificial intelligence-based analytical tool to identify those mCRC patients with high risk of disease progression and mortality based on their clinical parameters. Methods: Through www.projectdatasphere.org we accessed datasets of two randomised phase III trials including chemo-naïve (NCT00364013, n = 1183 patients) and chemo-refractory (NCT00113763, N = 483) mCRC patients. We generated synthetic fingerprints (SF) for each patient through the integration of 44 clinical features (demographics, anthropometrics, medical history, blood tests and treatment characteristics) collected, respectively, during the screening phase and the first month of inclusion in each trial. These SF were then input into a deep learning framework (DLF) to identify subgroup of patients based on their similarities. The resultant clusters were correlated with progression-free survival (PFS) and overall survival (OS). Results: After discarding missing data, 861 chemo-naïve and 341 chemo-resistant mCRC patients were eligible for the study. In the chemo-naïve cohort, the SF/DLF system was able to detect two different clusters: C0 (n = 31) and C1 (n = 830). Patients in C0 had a higher risk of progression (median PFS 6.2 months vs. 9.1 months; hazard ratio 1.83, 95% CI 1.16-2.88; p = 0.008) and death (median OS 13.2 months vs. 20.1 months; hazard ratio 2.84, 95% CI 1.68-4.80; p < 0.001) compared to patients in C1. When applied to the chemo-resistant cohort, the SF/DLF system was again able to identify two different clusters: P0 (n = 159) and P1 (n = 182). Patients in P0 had a higher risk of progression (median PFS 1.7 months vs. 1.8 months; hazard ratio 1.32, 95% CI 1.05-1.67; p < 0.001) and death (median OS 6.1 months vs. 6.8 months; hazard ratio 1.34, 95% CI 1.07-1.68; p = 0.01) compared to patients in P1. In both cases, feature contribution analysis showed that major differences between clusters were related to clinical status, anthropometrics and haematological and biochemistry tests. Conclusions: Our SF/DLF system can identify mCRC subtypes based on distinct clinical features that correlate with higher risk of progression and death. Further work is required to validate this approach as a novel prognostic biomarker tool for monitoring mCRC patients.

Trabectedin (Yondelis®, ET-743) is a marine-derived natural product that was initially isolated from the marine ascidian Ecteinascidia turbinata and is currently prepared synthetically. Trabectedin is used as a single agent for the treatment of patients with soft tissue sarcoma after failure of doxorubicin or ifosfamide or who are unsuited to receive these agents, and in patients with relapsed, platinum-sensitive ovarian cancer in combination with pegylated liposomal doxorubicin. Trabectedin presents a complex mechanism of action affecting key cell biology processes in tumor cells as well as in the tumor microenvironment. The inhibition of trans-activated transcription and the interaction with DNA repair proteins appear as a hallmark of the antiproliferative activity of trabectedin. Inhibition of active transcription is achieved by an initial direct mechanism that involves interaction with RNA polymerase II, thereby inducing its ubiquitination and degradation by the proteasome. This subsequently modulates the production of cytokines and chemokines by tumor and tumor-associated macrophages. Another interesting effect on activated transcription is mediated by the displacement of oncogenic transcription factors from their target promoters, thereby affecting oncogenic signaling addiction. In addition, it is well established that DNA repair systems including transcription-coupled nucleotide excision repair and homologous recombination play a role in the antitumor activity of trabectedin. Ongoing studies are currently addressing how to exploit these unique mechanistic features of trabectedin to combine this agent either with immunological or microenvironmental modulators or with classical chemotherapeutic agents in a more rational manner.

Background: In order to investigate the mechanisms of acquired resistance to trabectedin, trabectedin-resistant human myxoid liposarcoma (402-91/T) and ovarian carcinoma (A2780/T) cell lines were derived and characterised in vitro and in vivo. Methods: Resistant cell lines were obtained by repeated exposures to trabectedin. Characterisation was performed by evaluating drug sensitivity, cell cycle perturbations, DNA damage and DNA repair protein expression. In vivo experiments were performed on A2780 and A2780/T xenografts. Results: 402-91/T and A2780/T cells were six-fold resistant to trabectedin compared with parental cells. Resistant cells were found to be hypersensitive to UV light and did not express specific proteins involved in the nucleotide excision repair (NER) pathway: XPF and ERCC1 in 402-91/T and XPG in A2780/T. NER deficiency in trabectedin-resistant cells was associated with the absence of a G2/M arrest induced by trabectedin and with enhanced sensitivity (two-fold) to platinum drugs. In A2780/T, this collateral sensitivity, confirmed in vivo, was associated with an increased formation of DNA interstrand crosslinks. Conclusions: Our finding that resistance to trabectedin is associated with the loss of NER function, with a consequent increased sensitivity to platinum drugs, provides the rational for sequential use of these drugs in patients who have acquired resistance to trabectedin.

Lurbinectedin is an inhibitor of active transcription of protein-coding genes, causing DNA-break accumulation, apoptosis and modulation of the tumor microenvironment. Early-phase clinical trials indicate promising activity of lurbinectedin in small-cell lung cancer. Here, we describe the rationale and design of ATLANTIS (NCT02566993), an open-label, randomized, multicenter Phase III study to compare the efficacy of lurbinectedin and doxorubicin combination with standard-of-care chemotherapy, investigator's choice of cyclophosphamide/doxorubicin/vincristine or topotecan, in patients with small-cell lung cancer that has progressed following one line of platinum-based chemotherapy. Patients are randomized in a 1:1 ratio. The primary end point is overall survival and key secondary end points include progression-free survival, best tumor response and duration of response, each assessed by independent review committee.

Background: Through several not-fully-characterised moonlighting functions, translation elongation factor eEF1A2 is known to provide a fitness boost to cancer cells. Furthermore, eEF1A2 has been demonstrated to confer neoplastic characteristics on preneoplastic, nontumourigenic precursor cells. We have previously shown that eEF1A2 is the target of plitidepsin, a marine drug currently in development for cancer treatment. Herein, we characterised a new signalling pathway through which eEF1A2 promotes tumour cell survival. Methods: Previously unknown binding partners of eEF1A2 were identified through co-immunoprecipitation, high-performance liquid chromatography-mass spectrometry and proximity ligation assay. Using plitidepsin to release eEF1A2 from those protein complexes, their effects on cancer cell survival were analysed in vitro. Results: We uncovered that double-stranded RNA-activated protein kinase (PKR) is a novel eEF1A2-interacting partner whose pro-apoptotic effect is hindered by the translation factor, most likely through sequestration and inhibition of its kinase activity. Targeting eEF1A2 with plitidepsin releases PKR from the complex, facilitating its activation and triggering a mitogen-activated protein kinase signalling cascade together with a nuclear factor-κB-dependent activation of the extrinsic apoptotic pathway, which lead to tumour cell death. Conclusions: Through its binding to PKR, eEF1A2 provides a survival boost to cancer cells, constituting an Achilles heel that can be exploited in anticancer therapy., We thank Doctors César de Haro and Juan José Berlanga for fruitful discussions and for providing essential reagents. We also thank Rafael Sánchez for his technical assistance. The proteomic analysis was performed in the Proteomics Unit of Complutense University of Madrid that belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. F.G. is the recipient of a Spanish MEC/MICINN (SAF2015-64629- C2-2-R) grant.

Background: Juvenile myelomonocytic leukaemia (JMML) and chronic myelomonocytic leukaemia (CMML) are myelodysplastic myeloproliferative (MDS/MPN) neoplasms with unfavourable prognosis and without effective chemotherapy treatment. Trabectedin is a DNA minor groove binder acting as a modulator of transcription and interfering with DNA repair mechanisms; it causes selective depletion of cells of the myelomonocytic lineage. We hypothesised that trabectedin might have an antitumour effect on MDS/MPN. Methods: Malignant CD14+ monocytes and CD34+ haematopoietic progenitor cells were isolated from peripheral blood/bone marrow mononuclear cells. The inhibition of CFU-GM colonies and the apoptotic effect on CD14+ and CD34+ induced by trabectedin were evaluated. Trabectedin's effects were also investigated in vitro on THP-1, and in vitro and in vivo on MV-4-11 cell lines. Results: On CMML/JMML cells, obtained from 20 patients with CMML and 13 patients with JMML, trabectedin - at concentration pharmacologically reasonable, 1-5 nM - strongly induced apoptosis and inhibition of growth of haematopoietic progenitors (CFU-GM). In these leukaemic cells, trabectedin downregulated the expression of genes belonging to the Rho GTPases pathway (RAS superfamily) having a critical role in cell growth and cytoskeletal dynamics. Its selective activity on myelomonocytic malignant cells was confirmed also on in vitro THP-1 cell line and on in vitro and in vivo MV-4-11 cell line models. Conclusions: Trabectedin could be good candidate for clinical studies in JMML/CMML patients.

Trabectedin is the first marine-derived anti-neoplastic drug approved for the treatment of advanced soft tissue sarcoma and, in combination with pegylated liposomal doxorubicin, for the treatment of patients with relapsed platinum-sensitive ovarian cancer. From the beginning of its development, trabectedin showed some peculiar properties that clearly distinguished it from other anti-cancer drugs. In this mini-review, we will outline the current state of knowledge regarding the mode of action of trabectedin, which appears to represent a new class of anti-neoplastic drugs acting both on cancer cells and on the tumour microenvironment.

The prevailing paradigm states that cancer cells acquire multiple genetic mutations in oncogenes or tumor suppressor genes whose respective activation/up-regulation or loss of function serve to impart aberrant properties, such as hyperproliferation or inhibition of cell death. However, a tumor is now considered as an organ-like structure, a complex system composed of multiple cell types (e.g., tumor cells, inflammatory cells, endothelial cells, fibroblasts, etc.) all embedded in an inflammatory stroma. All these components influence each other in a complex and dynamic cross-talk, leading to tumor cell survival and progression. As the microenvironment has such a crucial role in tumor pathophysiology, it represents an attractive target for cancer therapy. In this review, we describe the mechanism of action of trabectedin and plitidepsin as an example of how these specific drugs of marine origin elicit their antitumor activity not only by targeting tumor cells but also the tumor microenvironment.

The dynamics of disease recurrence shows a bimodal pattern with a fairly broad dominant peak at about 1.5-2 years after surgery followed by a second peak at about 5 years. Nowadays, this clinical pattern is explained by assuming that primary breast tumours as well as their metastases have phases of both arrested (tumour dormancy) and active Gompertzian growth. Tumour dormancy at metastatic sites is currently ascribed to biological particularities of local tissue microenvironments that inhibit the growth of tumour cells. However, in some patients, tumour dormancy appears to also depend on the direct interplay between the primary tumour and those metastases, a biological phenomenon called "concomitant resistance". Concomitant resistance is related to three biological processes: concomitant immunity, tumour-induced angiogenesis and athrepsia. Concomitant resistance can explain the bimodal relapse pattern of breast cancer patients as well as many other clinical phenomena such as the better clinical outcome among patients surgically treated during the putative early luteal phase, or the worse clinical outcome of African-American premenopausal women. Any therapeutic interventions (even surgery) can affect concomitant resistance with the potential to induce a worse as well as a better clinical outcome. This should be taken into account when planning new treatment strategies.

Background and Purpose We have previously shown that cells with a defective Fanconi anaemia (FA) pathway are hypersensitive to trabectedin, a DNA-binding anti-cancer tetrahydroisoquinoline (DBAT) whose adducts functionally mimic a DNA inter-strand cross link (ICL). Here we expand these observations to new DBATs and investigate whether our findings in primary untransformed cells can be reproduced in human cancer cells. Experimental Approach Initially, the sensitivity of transformed and untransformed cells, deficient or not in one component of the FA pathway, to mitomycin C (MMC) and three DBATs, trabectedin, Zalypsis and PM01183, was assessed. Then, the functional interaction of these drugs with the FA pathway was comparatively investigated. Key Results While untransformed FA-deficient haematopoietic cells were hypersensitive to both MMC and DBATs, the response of FA-deficient squamous cell carcinoma (SCC) cells to DBATs was similar to that of their respective FA-competent counterparts, even though these FA-deficient SCC cells were hypersensitive to MMC. Furthermore, while MMC always activated the FA pathway, the DBATs inhibited the FA pathway in the cancer cell lines tested and this enhanced their response to MMC. Conclusions and Implications Our data show that although DBATs functionally interact with DNA as do agents that generate classical ICL, these drugs should be considered as FA pathway inhibitors rather than activators. Moreover, this effect was most significant in a variety of cancer cells. These inhibitory effects of DBATs on the FA pathway could be exploited clinically with the aim of ‘fanconizing’ cancer cells in order to make them more sensitive to other anti-tumour drugs.

This study: (i) investigated the in vitro cytotoxicity and mode of action of lurbinectedin (PM01183) and Zalypsis® (PM00104) compared with trabectedin in cell lines deficient in specific mechanisms of repair, (ii) evaluated their in vivo antitumor activity against a series of murine tumors and human xenografts. The antiproliferative activity, the DNA damage and the cell cycle perturbations induced by the three compounds on tumor lines were very similar. Nucleotide Excision Repair (NER) deficient cells were approximately fourfold more resistant to trabectedin, lurbinectedin and Zalypsis®. Cells deficient in non-homologous end joining (NHEJ), MRN complex and translesion synthesis (TLS) were slightly more sensitive to the three compounds (approximately fivefold) while cells deficient in homologous recombination (HR) were markedly more sensitive (150–200-fold). All three compounds showed a good antitumor activity in several in vivo models. Lurbinectedin and trabectedin had a similar pattern of antitumor activity in murine tumors and in xenografts, whereas Zalypsis® appeared to have a distinct spectrum of activity. The fact that no relationship whatsoever was found between the in vitro cytotoxic potency and the in vivo antitumor activity, suggests that in addition to direct cytotoxic mechanisms other host-mediated effects are involved in the in vivo pharmacological effects.

Elisidepsin (PM02734, Irvalec®) is a synthetic marine-derived cyclic peptide of the Kahalalide F family currently in phase II clinical development. Elisidepsin was shown to induce rapid oncosis in ErbB3-expressing cells. Other predictive factors of elisidepsin sensitivity remained unknown. A panel of 23 cancer cell lines of different origin was assessed for elisidepsin cytotoxicity and correlated with mutational state, mRNA and protein expression of selected genes. Elisidepsin showed potent and broad cytotoxic effects in our cancer cell line panel, being active at concentrations ranging from 0.4 to 2 μM that may be relevant for clinical settings. We have shown that elisidepsin is more active in cells harboring epithelial phenotype with high E-cadherin and low vimentin expression. In addition, high ErbB3 and Muc1 expression was correlated with sensitivity to elisidepsin, whereas the presence of KRAS activating mutations was associated with resistance. In DU-PM cells with acquired resistance to elisidepsin, ErbB3 expression was decreased, while Bcl2 was increased. DU-PM cells displayed higher sensitivity to ErbB1-inhibitors suggesting possible cross-talk of ErbB1 and ErbB3 signaling pathways. Combinations of elisidepsin with lapatinib and several chemotherapies including 5-FU and oxaliplatin resulted in synergistic effects that offer the potential of clinical use of elisidepsin in combination settings.

We have defined the mechanism of action of lurbinectedin, a marine-derived drug exhibiting a potent antitumor activity across several cancer cell lines and tumor xenografts. This drug, currently undergoing clinical evaluation in ovarian, breast, and small cell lung cancer patients, inhibits the transcription process through (i) its binding to CG-rich sequences, mainly located around promoters of protein-coding genes; (ii) the irreversible stalling of elongating RNA polymerase II (Pol II) on the DNA template and its specific degradation by the ubiquitin/proteasome machinery; and (iii) the generation of DNA breaks and subsequent apoptosis. The finding that inhibition of Pol II phosphorylation prevents its degradation and the formation of DNA breaks after drug treatment underscores the connection between transcription elongation and DNA repair. Our results not only help to better understand the high specificity of this drug in cancer therapy but also improve our understanding of an important transcription regulation mechanism. Mol Cancer Ther; 15(10); 2399–412. ©2016 AACR.

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.

eEF1A2 is one of the isoforms of the alpha subunit of the eukaryotic Elongation Factor 1. It is overexpressed in human tumors and is endowed with oncogenic properties, favoring tumor cell proliferation while inhibiting apoptosis. We demonstrate that plitidepsin, an antitumor agent of marine origin that has successfully completed a phase-III clinical trial for multiple myeloma, exerts its antitumor activity by targeting eEF1A2. The drug interacts with eEF1A2 with a K D of 80 nM and a target residence time of circa 9 min. This protein was also identified as capable of binding [ 14 C]-plitidepsin in a cell lysate from K-562 tumor cells. A molecular modelling approach was used to identify a favorable binding site for plitidepsin at the interface between domains 1 and 2 of eEF1A2 in the GTP conformation. Three tumor cell lines selected for at least 100-fold more resistance to plitidepsin than their respective parental cells showed reduced levels of eEF1A2 protein. Ectopic expression of eEF1A2 in resistant cells restored the sensitivity to plitidepsin. FLIM-phasor FRET experiments demonstrated that plitidepsin localizes in tumor cells sufficiently close to eEF1A2 as to suggest the formation of drug-protein complexes in living cells. Altogether, our results strongly suggest that eEF1A2 is the primary target of plitidepsin.

Trabectedin (ET-743) is a marine alkaloid isolated from the Caribbean tunicate Ecteinascidia turbinata, with a chemical structure characterized by three fused tetrahydroisoquinoline rings. Two of these rings (subunits A and B) provide the framework for covalent interaction with the minor groove of the DNA double helix, whereas the third ring (subunit C) protrudes from the DNA duplex, apparently allowing interactions with adjacent nuclear proteins. The compound's chemical interactions trigger a cascade of events that interfere with several transcription factors, DNA binding proteins, and DNA repair pathways, likely to be different from other DNA-interacting agents. Trabectedin also causes modulation of the production of cytokines and chemokines by tumor and normal cells, suggesting that the antitumor activity could also be ascribed to changes in the tumor microenvironment. The promising data on the combination of trabectedin with other anticancer agents, observed in preclinical systems, have prompted several clinical studies that are currently ongoing. One of these combinations (trabectedin-pegylated liposomal doxorubicin) was recently authorized by the European Commission for the treatment of patients with relapsed platinum-sensitive ovarian cancer. Mol Cancer Ther; 9(8); 2157–63. ©2010 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: Plitidepsin (APL), an antitumor agent isolated from the marine tunicate Aplidium albicans, has been tested with positive results in multiple myeloma (MM) patients in a phase III pivotal trial in combination with dexamethasone (clinicaltrials.gov identifier: NCT01102426) and in a phase I trial in combination with bortezomib and dexamethasone (clinicaltrials.gov identifier: NCT02100657). Plitidepsin targets eEF1A2, one of two isoforms of the alpha subunit of the eEF1 complex. In mammals, eEF1A2 has a selective pattern of expression in those tissues that do not express the A1 isoform, namely brain and muscle. Nonetheless, eEF1A2 is aberrantly expressed in many cancers, including solid tumors (1-3) and MM (4), and has been shown to hold oncogenic properties (5). We have previously demonstrated through several means the interaction of plitidepsin with its target, eEF1A2 and calculated a Kd of around 80 nM for this interaction (6). Indeed, we have found that plitidepsin exclusively binds to the GTP-bound form of eEF1A2 Aims: Here we investigated whether eEF1A2 levels had any effect on the sensitivity of MM cells to both plitidepsin and bortezomib, to better understand the synergistic effect these two drugs yield when combined (7). Methods: RPMI-8226, OPM1, U266-B1, MM1S and NCI-NH929 multiple myeloma cells were cultured in optimal conditions. eEF1A2 (GTX102326) and autophagy (L7543) were followed by Western blot. Dose-response experiments were performed in 96 well plates, treating cells with increasing doses of the compounds for 24 hours and measuring cell growth with MTT. Curves were then fitted with GraphPad Prism5 software. For combination experiments, the Chou-Talalay procedure was used. Results: We first analyzed the level of eEF1A2 protein in a panel of multiple myeloma cell lines. Then, we selected a cell line with low expression, MM-1S, and one with high expression, OPM-1, of the elongation factor and checked the sensitivity to both, bortezomib and plitidepsin through dose-response experiments. Interestingly, the overexpression of eEF1A2, the target of plitidepsin, seems to be related to an increased resistance to bortezomib, a phenomenon usually related to enhanced autophagy since this latter pathway compensates the inhibition of proteasome. eEF1A, in its GTP-bound form, has been shown to inhibit chaperone-mediated autophagy (CMA), essential for the degradation of unfolded proteins (8). Since plitidepsin binds to the GTP-bound eEF1A2, probably stabilizing this form of the elongation factor, it seems likely that, in such a way, it may inhibit CMA. We explored this possibility and, indeed, we observed that plitidepsin inhibited autophagy in MM cells. Summary/Conclusion: Plitidepsin treatment presumably counteracts the main mechanism of acquired resistance to bortezomib, namely autophagy. (1)Sun et al., 2014, Biochem Biophys Res Commun 450:1-6. (2)Xu et al., 2013, Clin Exp Metastasis 30:933-44. (3)Anand et al., 2002, Nat Genet 31:301-5. (4)Li et al., 2010, PLOS One 5, e10755. (5)Lee and Surh, 2009, Ann N Y Acad Sci 1171:87-93. (6)Losada et al., 2004, Br J Cancer 91:1405-13. (7)Mitsiades et al., 2008, Cancer Res 68:5216-25. (8)Bandyopadhyay et al., 2010, Mol Cel 39:535-47. Citation Format: Juan F. Martínez-Leal, Gaëlle Quiniou, Alejandro Losada, María José Muñoz, Rafael Sanchez-Mesa, Patricia Martinez-Rivas, Juan Manuel Dominguez, Carlos M. Galmarini. Plitidepsin inhibits autophagy, the main mechanism of acquired resistance to bortezomib [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B057.

Purpose: To evaluate the role of microtubule-associated variables as potential predictors of response and clinical outcome in patients with advanced breast cancer receiving single-agent docetaxel or doxorubicin chemotherapy.Experimental Design: The analysis was done on 173 tumor samples from patients with locally advanced or metastatic breast cancer who have participated in the TAX-303 phase III trial in which patients were randomly assigned to receive docetaxel or doxorubicin. Expression of total α- and β-tubulin, classes II to IV β-tubulin isotypes, and τ protein was evaluated by immunohistochemistry on formalin-fixed, paraffin-embedded tumors from the primary breast cancer.Results: We observed that patients with “high” expression of class III β-tubulin isotype had a higher probability of response to docetaxel than to doxorubicin treatment (odds ratio, 1.9; 95% confidence interval, 1.01-3.7; P = 0.05). No difference was observed in terms of time to progression or in terms of overall survival.Conclusions: This study suggests that the superiority of docetaxel over doxorubicin seems to be confined to the subgroup of patients with “high” expression of class III β-tubulin isotype.

Despite its increasing clinical use, almost no data are currently available about paclitaxel effects on non-cancerous mammary epithelial cells. We have previously established paclitaxel-resistant sub-cell lines (paclitaxel-surviving populations, PSPs; n=20), and sensitive controls (control clones, CCs; n=10), from the untransformed human mammary epithelial cell line HME1. In this study, we aimed to establish whether paclitaxel resistance was associated with a modified sensitivity to paclitaxel-induced aneuploidy. For this purpose, we analysed basal and paclitaxel-induced chromosome missegregation, apoptosis and aberrant spindle multipolarisation as well as microtubular network composition for each subline. PSP sublines showed higher basal and paclitaxel-induced chromosome missegregation than the CC sublines. This phenomenon was associated with resistance to paclitaxel-induced apoptosis. No significant difference in paclitaxel-induced spindle pole abnormalities between CC and PSP sublines was found. Besides, we showed that a majority of PSPs display a constitutively disrupted microtubular network composition due to aberrant tubulin expression and post-translational modifications. These results clearly indicate that paclitaxel resistance in untransformed human mammary epithelial cells is related to an increased susceptibility to acquire aneuploidy in response to this agent. The consequences of these paclitaxel-associated alterations could be deleterious as they can potentially trigger tumorigenesis.

Resistance of human tumors to anticancer drugs is most often ascribed to gene mutations, gene amplification, or epigenetic changes that influence the uptake, metabolism, or export of drugs from single cells. Another important yet little-appreciated cause of anticancer drug resistance is the limited ability of drugs to penetrate tumor tissue and to reach all of the tumor cells in a potentially lethal concentration. To reach all viable cells in the tumor, anticancer drugs must be delivered efficiently through the tumor vasculature, cross the vessel wall, and traverse the tumor tissue. In addition, heterogeneity within the tumor microenvironment leads to marked gradients in the rate of cell proliferation and to regions of hypoxia and acidity, all of which can influence the sensitivity of the tumor cells to drug treatment. In this review, we describe how the tumor microenvironment may be involved in the resistance of solid tumors to chemotherapy and discuss potential strategies to improve the effectiveness of drug treatment by modifying factors relating to the tumor microenvironment.

This study assessed the antiproliferative activity of sapacitabine (CYC682, CS-682) in a panel of 10 human cancer cell lines with varying degrees of resistance or sensitivity to the commonly used nucleoside analogues ara-C and gemcitabine. Growth inhibition studies using sapacitabine and CNDAC were performed in the panel of cell lines and compared with both nucleoside analogues and other anticancer compounds including oxaliplatin, doxorubicin, docetaxel and seliciclib. Sapacitabine displayed antiproliferative activity across a range of concentrations in a variety of cell lines, including those shown to be resistant to several anticancer drugs. Sapacitabine is biotransformed by plasma, gut and liver amidases into CNDAC and causes cell cycle arrest predominantly in the G(2)/M phase. No clear correlation was observed between sensitivity to sapacitabine and the expression of critical factors involved in resistance to nucleoside analogues such as deoxycytidine kinase (dCK), human equilibrative nucleoside transporter 1, cytosolic 5'-nucleotidase and DNA polymerase-alpha. However, sapacitabine showed cytotoxic activity against dCK-deficient L1210 cells indicating that in some cells, a dCK-independent mechanism of action may be involved. In addition, sapacitabine showed a synergistic effect when combined with gemcitabine and sequence-specific synergy with doxorubicin and oxaliplatin. Sapacitabine is therefore a good candidate for further evaluation in combination with currently used anticancer agents in tumour types with unmet needs.

We investigated the potential in vivo aneugenic effects associated with paclitaxel treatment. For this purpose, we treated female nude mice with paclitaxel using doses equivalent to those used in weekly schedules at the clinical level (three cycles of 30 mg/kg/week for three consecutive weeks followed by one resting week). We then evaluated the frequencies of micronucleated erythrocytes (MNE) in peripheral blood using the acridine orange micronucleus assay. The frequency of MNE was evaluated after 24 h and 168 h of administration of the last dose of each paclitaxel cycle (STA mice group) as well as after one year of the first dose of treatment (LTA mice group). We also analyzed the cytology of peripheral blood and bone marrows obtained from these mice at each time period. In the STA mice group, three cycles of paclitaxel induced a 2.4-fold increase in MNE frequencies compared to the control group (p0.01). This effect was observed after 24 h of the last dose of each chemotherapy cycle and persisted at least for 168 h. In the LTA mice group, paclitaxel-treated mice presented a 1.8-fold increase in the MNE frequency (p = 0.01) indicating that paclitaxel-induced MNE increase lasted for at least one year. Although the appearance of micronuclei in erythrocytes and granulocytes in peripheral blood and bone marrow cytological smears, there was no evidence of myeloproliferative disease. The present data therefore indicate an aneugenic potential of paclitaxel for humans, which should be considered in the risk-benefit analysis of its increasing clinical use.

Purpose Trabectedin induces synthetic lethality in tumor cells carrying defects in homologous recombinant DNA repair. We evaluated the effect of concomitant inhibition of nucleotide-excision repair and poly (ADP-ribose) polymerase (PARP) activity with trabectedin and PARP inhibitors, respectively, and whether the synthetic lethality effect had the potential for a synergistic effect in breast cancer cell lines. Additionally, we investigated if this approach remained effective in BRCA1-positive breast tumor cells. Methods We have evaluated the in vitro synergistic effect of combinations of trabectedin and three different PARP inhibitors (veliparib, olaparib, and iniparib) in four breast cancer cell lines, each presenting a different BRCA1 genetic background. Antiproliferative activity, DNA damage, cell cycle perturbations and poly(ADP-ribosyl)ation were assessed by MTT assay, comet assay, flow cytometry and western blot, respectively. Results The combination of trabectedin and olaparib was synergistic in all the breast cancer cell lines tested. Our data indicated that the synergy persisted regardless of the BRCA1 status of the tumor cells. Combination treatment was associated with a strong accumulation of double-stranded DNA breaks, G2/M arrest, and apoptotic cell death. Synergistic effects were not observed when trabectedin was combined with veliparib or iniparib. Conclusion Collectively, our results indicate that the combination of trabectedin and olaparib induces an artificial synthetic lethality effect that can be used to kill breast cancer cells, independent of BRCA1 status.

Producción Científica, Recent preclinical evidence has suggested that Ewing Sarcoma (ES) bearing EWSR1-ETS fusions could be particularly sensitive to PARP inhibitors (PARPinh) in combination with DNA damage repair (DDR) agents. Trabectedin is an antitumoral agent that modulates EWSR1-FLI1 transcriptional functions, causing DNA damage. Interestingly, PARP1 is also a transcriptional regulator of EWSR1-FLI1, and PARPinh disrupts the DDR machinery. Thus, given the impact and apparent specificity of both agents with regard to the DNA damage/DDR system and EWSR1-FLI1 activity in ES, we decided to explore the activity of combining PARPinh and Trabectedin in in vitro and in vivo experiments. The combination of Olaparib and Trabectedin was found to be highly synergistic, inhibiting cell proliferation, inducing apoptosis, and the accumulation of G2/M. The drug combination also enhanced γH2AX intranuclear accumulation as a result of DNA damage induction, DNA fragmentation and global DDR deregulation, while EWSR1-FLI1 target expression remained unaffected. The effect of the drug combination was corroborated in a mouse xenograft model of ES and, more importantly, in two ES patient-derived xenograft (PDX) models in which the tumors showed complete regression. In conclusion, the combination of the two agents leads to a biologically significant deregulation of the DDR machinery that elicits relevant antitumor activity in preclinical models and might represent a promising therapeutic tool that should be further explored for translation to the clinical setting., Ministerio de Economía y Competitividad (PI081828), Ministerio de Economía y Competitividad (RD06/0020/0059 ), Ministerio de Economía y Competitividad (RD12/0036/0017), Ministerio de Economía y Competitividad (PT13/0010/0056)

Cancers arise from the sequential acquisition of genetic alterations in specific genes. The high number of mutations in cancer cells led to the hypothesis that an early step in tumor progression is the generation of a genetic instability. The potent role of genetic instability in initiation and progression of colorectal cancers has been well defined in hereditary nonpolyposis colon cancer (HNPCC) syndrome. HNPCC is a common hereditary disorder caused by germline mutations of DNA mismatch repair (MMR) genes. Somatic loss of the normal allele of the predisposition gene leads to a strong "mutator phenotype", characterized by a high rate of mutations in repetitive sequences. Nevertheless, the observation of frequent alterations of key growth regulatory genes in MMR-deficient cells such as NF1, APC, p53, K-Ras, with no significant excess of frameshift mutations and changes at short coding repeats, suggest that even in the presence of an inherited tendency to genomic instability, tumor progression is mainly driven by a process of natural selection.

In any disease, the knowledge of the natural history of untreated cases provides a real background against which the real advantages of a new treatment itself are judged. Fortunately, in the present days, there are scant data on outcomes in patients with untreated breast cancer. In an attempt to provide this background against which the virtues of current curative and palliative treatments can be more accurately assessed, we have reviewed the literature regarding published untreated breast cancer series. Taking into consideration all the difficulties of analyzing reports written on the last half of the nineteenth century or on the first half of the twentieth century, in most reports, patients survived almost 3–4 years without any type of treatment. Worth mentioning, approximately 5–10 % of untreated patients lived longer than 10 years. Thus, the spectrum of clinical aggressiveness of untreated breast cancer varies between virulence and chronic disease. These facts should be taken into account when considering the value of current treatments for early-stage disease.

Acute myelogenous leukemia (AML) is the most common form of acute leukemia in adults. After diagnosis, patients with AML are mainly treated with standard induction chemotherapy combining cytarabine (araC) and anthracyclines. The majority of them achieve complete remission (CR) (65-80%). However, prospects for long-term survival are poor for the majority of patients. Resistance to chemotherapy therefore remains a major obstacle in the effective treatment of patients with AML. In this review, we highlight the current knowledge of substrate cycles involved in normal deoxynucleoside triphosphate (dNTPs) metabolism and their possible role in drug resistance to araC. © 2005 Taylor & Francis Group Ltd. Fil: Fernandez Calotti, Paula. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Jordheim, Lars Petter. Université Claude Bernard Lyon 1; Francia Fil: Giordano, Mirta Nilda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; Argentina Fil: Dumontet, Charles. Université Claude Bernard Lyon 1; Francia Fil: Galmarini, Carlos Maria. Université Claude Bernard Lyon 1; Francia

Resistance to cytotoxic nucleoside analogues is a major problem in cancer treatment. The cellular mechanisms involved in this phenomenon have been studied for several years, and some factors have been identified. Various strategies to overcome resistance have been suggested, but none has yet shown efficacy in vivo. We developed a gemcitabine-resistant cell line (L1210 10K) from the murine leukemic L1210 strain (L1210 wt) by continuous exposure to increasing concentrations of gemcitabine. L1210 10K is highly resistant to gemcitabine (14,833-fold), 1-β-d-arabinofuranosylcytosine (ara-C; 2,100-fold), troxacitabine (>200-fold), and cladribine (160-fold) and slightly resistant to trimidox (7.22-fold), but does not display cross-resistance to fludarabine or nonnucleoside anticancer drugs. Deoxycytidine kinase mRNA was not detected by quantitative real-time reverse transcription-PCR in L1210 10K cells, whereas expression of thymidine kinase 1 and ribonucleotide reductase subunit R2 gene was moderately reduced. L1210 10K cells also demonstrated in vivo resistance to nucleoside analogues: gemcitabine- or ara-C-treated mice carrying L1210 10K had significantly shorter survival than gemcitabine- or ara-C-treated mice carrying L1210 wt (P < 0.05). UA911, a mononucleotide prodrug (pronucleotide) of ara-C was found to significantly sensitize L1210 10K cells in vitro. These results suggest that reduced deoxycytidine kinase expression is a mechanism of resistance to gemcitabine that is relevant in vivo and can be circumvented by a prodrug approach.

B-cell chronic lymphocytic leukemia (B-CLL) is a malignant disease characterized by an accumulation of monoclonal CD5+ mature B cells, with a high percentage of cells arrested in the G0/G1 phase of the cell cycle, and a particular resistance toward apoptosis-inducing agents. Dok1 (downstream of tyrosine kinases) is an abundant Ras-GTPase-activating protein (Ras-GAP)-associated tyrosine kinase substrate, which negatively regulates cell proliferation, downregulates MAP kinase activation and promotes cell migration. The gene encoding Dok1 maps to human chromosome 2p13, a region previously found to be rearranged in B-CLL. We have screened the Dok1 gene for mutations from 46 individuals with B-CLL using heteroduplex analysis. A four-nucleotide GGCC deletion in the coding region was found in the leukemia cells from one patient. This mutation causes a frameshift leading to protein truncation at the carboxyl-terminus, with the acquisition of a novel amino-acid sequence. In contrast to the wild-type Dok1 protein, which has cytoplasmic/membrane localization, the mutant Dok1 is a nuclear protein containing a functional bipartite nuclear localization signal. Whereas overexpression of wild-type Dok1 inhibited PDGF-induced MAP kinase activation, this inhibition was not observed with the mutant Dok1. Furthermore the mutant Dok1 forms heterodimers with Dok1 wild type and the association can be enhanced by Lck-mediated tyrosine-phosphorylation. This is the first example of a Dok1 mutation in B-CLL and the data suggest that Dok1 might play a role in leukemogenesis.

Adequate intracellular concentrations of ara-CMP, the monophosphorylated derivative of ara-C, are essential for its cytotoxicity. The critical step for ara-CMP formation is intracellular phosphorylation of ara-C by deoxycytidine kinase (dCK). A common nucleoside resistance mechanism is mutation affecting the expression or the specificity of dCK. We describe the ability of a tert-butyl S-acyl-thioethyl (SATE) derivative of ara-CMP (UA911) to circumvent ara-C resistance in a dCK-deficient human follicular lymphoma cell line (RL-G). The RL-G cell line was produced by continuous exposure to gemcitabine and displayed low dCK mRNA and protein expression that conferred resistance both to ara-C (2,250-fold) and to gemcitabine (2,092-fold). RL-G cells were able to take up the UA911 pronucleotide by diffusion and metabolize it to the corresponding ara-CMP and ara-CTP nucleotides, exhibiting a 199-fold reduction in resistance ratios, and a similar cell cycle arrest to the parental RL-7 cells. Exposures to 10, 50 or 100 microM concentrations of UA911 produced 160 +/- 7, 269 +/- 8 and 318 +/- 62 pmol ara-CTP/mg protein in RL-7 cells, and 100 +/- 12, 168 +/- 10 and 217 +/- 39 pmol ara-CTP/mg protein in RL-G cells, respectively. Exposure of RL-G cells to underivatized, radiolabeled ara-C produced no detectable amounts of the active triphosphate metabolites. We conclude that the UA911 pronucleotide is capable of overcoming dCK-mediated resistance. This result can be attributed to the unique cellular metabolism of the SATE pronucleotides giving rise to the intracellular delivery of ara-CMP to dCK-deficient cells.

Cytotoxic nucleoside analogs (NA) are important in the treatment of hematologic malignancies. The NA in routine clinical use include the pyrimidine analog cytosine arabinoside (ara-c), which is extensively used in the treatment of acute leukemias, and the purine analogs, cladribine and fludarabine. These drugs have mostly been used in the treatment of low grade hematological malignancies. NA become therapeutically effective only after phosporylation to the triphosphate level. The 5'-nucleotidases (5'-NTs) dephosphorylate the monophosphate form of NA and, therefore, may affect the pharmacological activity of these antimetabolites in the clinic. Several 5'-NTs attached to membranes or present in the cytosol or in mitochondria are present in mammalian cells. cN-II, an IMP-selective 5'-NT, participates in the regulation of purine deoxyribonucleotide metabolism. cN-II opposes the action of the salvage enzymes by dephosphorylating purine nucleoside mononphosphates to purine nucleosides. Due to its phosphotransferase activity, cN-II can also phosphorylate inosine and 2',3'-dideoxyribonucleosides utilizing IMP as a phosphate donor. The observation that cytosolic cN-II is able to phosphorylate purine nucleosides has initiated studies on its potential participation in the metabolism of anticancer agents and in the development of cN-II inhibitory substances. In this review, we highlight the current knowledge concerning cN-II activity and regulation of intracellular deoxyribonucleotide pools and it role in hematological malignancies.

The cytotoxic activity of cytarabine (ara-C) in leukaemic blasts depends on activating enzymes such as deoxycytidine kinase (dCK) and inactivating enzymes such as the 5'-nucleotidases. We have analysed dCK and 'high-Km' 5'-nucleotidase (cN-II) mRNA expression by the quantitative real-time polymerase chain reaction at diagnosis in leukaemic blasts from 115 acute myeloid leukaemia (AML) patients treated with ara-C. The prognostic value of these parameters as well as that of the cN-II/dCK ratio was determined. In univariate analyses: (1) low levels of dCK, high levels of cN-II and a high cN-II/dCK ratio predicted shorter disease-free survival (DFS); (2) low levels of dCK and cN-II/dCK ratio also predicted shorter overall survival (OS). In a multivariate analysis taking into account other clinical and laboratory variables: (1) high cN-II expression, a high cN-II/dCK ratio, age >/= 60 years and an unfavourable karyotype were independent prognostic factors for DFS; and (2) a high cN-II/dCK ratio, age >/= 60 years and an unfavourable karyotype predicted shorter OS. Age, karyotype and cN-II/dCK ratio were used to define a prognostic score that permitted the identification of high- and low-risk groups. Our results suggest that dCK and cN-II mRNA expression in leukaemic blasts at diagnosis is correlated with clinical outcome and may play a functional role in the resistance to ara-C in patients with AML.

In the present study, we compared the dynamics and composition of microtubules in cell lines derived from the human breast adenocarcinoma MCF-7 containing either the wild-type p53 (wt-p53; MN1) or a dominant-negative variant of p53 gene (mut-p53; MDD2). Mut-p53 cells were significantly resistant to the cytotoxicity of the microtubule-targeted drugs (vinca alkaloids and taxanes), as compared with wt-p53 cells. Studies by high-resolution time-lapse fluorescence microscopy in living cells indicated that the dynamics of microtubules of mut-p53 cells were altered in complex ways and were significantly increased as compared with microtubules in wt-p53 cells. The percentage of time microtubules spent in growing and shortening phases increased significantly, their catastrophe frequency increased, and their overall dynamicity increased by 33%. In contrast, their shortening rate and the mean length shortened decreased. Cells containing mut-p53 displayed increased polymerisation of tubulin, increased protein levels of the class IV beta-tubulin isotype, STOP and survivin, and reduced protein levels of class II beta-tubulin isotype, MAP4 and FHIT. We conclude that p53 protein may contribute to the regulation of microtubule composition and function, and that alterations in p53 function may generate complex microtubule-associated mechanisms of resistance to tubulin-binding agents.

Factors that reduce the intracellular concentration of triphosphorylated cytarabine (ara-CTP), the active form of cytarabine (ara-C), may induce chemoresistance in acute myeloid leukaemia (AML) patients. These factors include reduced influx of ara-C by the hENT1 transporter, reduced phosphorylation by deoxycytidine kinase (dCK), and increased degradation by high Km cytoplasmic 5'-nucleotidase (5NT) and/or cytidine deaminase (CDD). Increased levels of DNA polymerase alpha (DNA POL) and reduced levels of topoisomerase I (TOPO I) and topoisomerase II (TOPO II) have also been detected in ara-C-resistant cell lines. To determine whether these factors are implicated in clinical ara-C resistance, we analysed the expression of these parameters at diagnosis, using reverse transcription polymerase chain reaction, in the blast cells of 123 AML patients treated with ara-C. At diagnosis, hENT1, dCK, CDD, 5NT, TOPO I, TOPO II, DNA POL and MDR1 were expressed in 83%, 22%, 7%, 37%, 59%, 37%, 39% and 16% of patients respectively. In univariate analysis, patients with expression of 5NT or DNA POL at diagnosis had significantly shorter disease-free survival (DFS). In multivariate analysis, DNA POL positivity and hENT1 deficiency were related to a shorter DFS. In univariate analysis, patients with 5NT-positive blasts had significantly shorter overall survival (OS). In multivariate analysis, shorter OS was related to DNA POL positivity. These results suggest that expression of DNA POL, 5NT and hENT1 at diagnosis may be resistance mechanisms to ara-C in AML patients.

Background: P-glycoprotein (P-gp)–mediated chemoresistance plays an important role in drug resistance. Methods: We investigated if P-gp expression by cancer cells affects cell cytotoxicity and cell-cycle perturbations induced by six commonly used chemotherapeutic agents (doxorubicin, daunorubicin, mitoxantrone, vinblastine, paclitaxel, and colchicine). For this purpose, we used KB cell lines as a model and flow cytometric cell viability and drug-induced cell-cycle perturbation–based methods. We continuously cultured KB cell lines, in the presence of various anticancer agents and measured the cell-cycle kinetics and percentage of cell viability by flow cytometry at different time intervals. Results: The highly resistant cell line KB V-1 was significantly less susceptible to drug cytotoxicity than the sensitive cell line KB 3-1. KB V-1 cells cultured with different chemotherapy agents continued cell-cycle progression without any significant perturbation of cell compartment distribution. Addition of verapamil, by inhibiting P-gp, reversed resistance thereby increasing drug cytotoxicity and allowing the appearance of drug induced cell-cycle perturbations. Conclusions: We conclude that P-gp interferes with cell cytotoxicity and cell-cycle perturbations induced by anticancer agents. Flow cytometry can be used to conveniently detect P-gp–mediated chemoresistance by cancer cells as it allows the analysis of cell cytotoxicity and cell-cycle modifications after exposure to specific chemotherapeutic and reversal agents.

Gemcitabine is a relatively new agent with promising activity in solid tumors. Few data are available regarding mechanisms of resistance to gemcitabine downstream from the drug-target interaction. The present study was performed to gain insight into the role of p53 status on the cytotoxicity of gemcitabine on cancer cells. Drug sensitivity, drug metabolism, cell kinetics and drug-induced apoptosis were compared in 2 lines derived from the mammary adenocarcinoma MCF-7: the wildtype p53 (wt-p53) containing MN-1 cell line and, the MDD2 line containing a dominant negative variant of the p53 protein (mut-p53). The MDD2 cell line was significantly more resistant to gemcitabine cytotoxicity than the MN-1 cell line. The resistant phenotype could not be attributed to a defective gemcitabine activation/degradation pathway or altered levels of expression of intracellular targets. Although both cell lines exhibited p53 accumulation, MN-1 but not MDD2 cells, displayed p21(WAF1) induction after exposure to gemcitabine. Gemcitabine induced an S-phase arrest in both cell lines. A more pronounced block in G1 phase, however, was observed in MN1 cells. Exposure to gemcitabine induced a higher degree of apoptosis in MN-1 than in MDD2 cells. This corresponded with suppression of Bcl-2 and Bcl-X/L expression in wt-p53 cells exposed to gemcitabine whereas Bcl-2 levels remained stable and Bcl-X/L levels increased in mut-p53 cells exposed to gemcitabine. We conclude that the p53 status of cancer cells influences their sensitivity to gemcitabine cytotoxicity. Our evidence suggests that loss of p53 function leads to loss of cell cycle control and alterations in the apoptotic cascade, conferring resistance to gemcitabine in cancer cell lines displaying a mut-p53.

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