Lurbinectedin versus pegylated liposomal doxorubicin or topotecan in patients with platinum-resistant ovarian cancer: A multicenter, randomized, controlled, open-label phase 3 study (CORAIL).

OBJECTIVE: The randomized phase 3 CORAIL trial evaluated whether lurbinectedin improved progression-free survival (PFS) compared to pegylated liposomal doxorubicin (PLD) or topotecan in patients with platinum-resistant ovarian cancer. METHODS: Patients were randomly assigned (1:1) to lurbinectedin 3.2 mg/m2 1-h i.v. infusion q3wk (experimental arm), versus PLD 50 mg/m2 1-h i.v. infusion q4wk or topotecan 1.50 mg/m2 30-min i.v. infusion Days 1-5 q3wk (control arm). Stratification factors were PS (0 vs. ≥1), prior PFI (1-3 months vs. >3 months), and prior chemotherapy lines (1-2 vs. 3). The primary endpoint was PFS by Independent Review Committee in all randomized patients. This study was registered with ClinicalTrials.gov, NCT02421588. RESULTS: 442 patients were randomized: 221 in lurbinectedin arm and 221 in control arm (127 PLD and 94 topotecan). With a median follow-up of 25.6 months, median PFS was 3.5 months (95% CI, 2.1-3.7) in the lurbinectedin arm and 3.6 months (95% CI, 2.7-3.8) in the control arm (stratified log-rank p = 0.6294; HR = 1.057). Grade ≥ 3 treatment-related adverse events (AEs) were most frequent in the control arm: 64.8% vs. 47.9% (p = 0.0005), mainly due to hematological toxicities. The most common grade ≥ 3 AEs were: fatigue (7.3% of patients) and nausea (5.9%) with lurbinectedin; mucosal inflammation (8.5%) and fatigue (8.0%) in the control arm. CONCLUSIONS: The primary endpoint of improvement in PFS was not met. Lurbinectedin showed similar antitumor efficacy and was better tolerated than current standard of care in patients with platinum-resistant ovarian cancer.

Nibrin is a marker of clinical outcome in patients with advanced serous ovarian cancer treated in the phase III OVA-301 trial.

OBJECTIVE: This study investigated the relationship between 13 proteins involved in DNA damage and the outcomes of patients with recurrent ovarian cancer (ROC). PATIENTS AND METHODS: Immunohistochemistry staining was performed in 114 diagnostic samples from patients with serous ROC who participated in the OVA-301 study, which compared pegylated liposomal doxorubicin (PLD) with a combination of trabectedin plus PLD. Percentage of positive cells for every marker was calculated and correlated with overall response rate (ORR), progression-free survival (PFS) and overall survival (OS). RESULTS: A statistically significant correlation between high levels of nibrin and lower ORR (P=0.03), shorter PFS (P=0.007) and shorter OS (P=0.01) was observed. After stratification, in patients with platinum-sensitive disease treated with the combination of trabectedin plus PLD, high levels of nibrin correlated with lower ORR (P=0.01) and shorter PFS (P=0.02). A better clinical outcome (ORR, PFS and OS) was also associated to low levels of CHK2 in trabectedin plus PLD treated patients. No correlations were found in PLD-treated patients. According to the results of a multivariate analysis, there was a statistically significant correlation between high nibrin (P=0.001) and low BRCA2 levels (P=0.03) and a worse PFS, and between high nibrin levels and a worse OS (P=0.006). CONCLUSION: Our results indicate that high nibrin expression seems to be associated with a worse clinical outcome in serous ROC, particularly in patients treated with the combination trabectedin plus PLD. Prospective studies to determine clinical usefulness of nibrin as a possible biomarker in other series of patients with ROC are warranted.

Predictive factors of sensitivity to elisidepsin, a novel Kahalalide F-derived marine compound.

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.

CXCR4-independent rescue of the myeloproliferative defect of the Gata1low myelofibrosis mouse model by Aplidin.

The discovery of JAK2 mutations in Philadelphia-negative myeloproliferative neoplasms has prompted investigators to evaluate mutation-targeted treatments to restore hematopoietic cell functions in these diseases. However, the results of the first clinical trials with JAK2 inhibitors are not as promising as expected, prompting a search for additional drugable targets to treat these disorders. In this paper, we used the hypomorphic Gata1(low) mouse model of primary myelofibrosis (PMF), the most severe of these neoplasms, to test the hypothesis that defective marrow hemopoiesis and development of extramedullary hematopoiesis in myelofibrosis is due to insufficient p27(Kip1) activity and is treatable by Aplidin, a cyclic depsipeptide that activates p27(Kip1) in several cancer cells. Aplidin restored expression of Gata1 and p27(Kip1) in Gata1(low) hematopoietic cells, proliferation of marrow progenitor cells in vitro and maturation of megakaryocytes in vivo (reducing TGF-beta/VEGF levels released in the microenvironment by immature Gata1(low) megakaryocytes). Microvessel density, fibrosis, bone growth, and marrow cellularity were normal in Aplidin-treated mice and extramedullary hematopoiesis did not develop in liver although CXCR4 expression in Gata1(low) progenitor cells remained low. These results indicate that Aplidin effectively alters the natural history of myelofibrosis in Gata1(low) mice and suggest this drug as candidate for clinical evaluation in PMF.

Trabectedin Reveals a Strategy of Immunomodulation in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a B-cell neoplasia characterized by protumor immune dysregulation involving nonmalignant cells of the microenvironment, including T lymphocytes and tumor-associated myeloid cells. Although therapeutic agents have improved treatment options for CLL, many patients still fail to respond. Some patients also show immunosuppression. We have investigated trabectedin, a marine-derived compound with cytotoxic activity on macrophages in solid tumors. Here, we demonstrate that trabectedin induces apoptosis of human primary leukemic cells and also selected myeloid and lymphoid immunosuppressive cells, mainly through the TRAIL/TNF pathway. Trabectedin modulates transcription and translation of IL6, CCL2, and IFNα in myeloid cells and FOXP3 in regulatory T cells. Human memory CD8+ T cells downregulate PD-1 and, along with monocytes, exert in vivo antitumor function. In xenograft and immunocompetent CLL mouse models, trabectedin has antileukemic effects and antitumor impact on the myeloid and lymphoid cells compartment. It depletes myeloid-derived suppressor cells and tumor-associated macrophages and increases memory T cells. Trabectedin also blocks the PD-1/PD-L1 axis by targeting PD-L1+ CLL cells, PD-L1+ monocytes/macrophages, and PD-1+ T cells. Thus, trabectedin behaves as an immunomodulatory drug with potentially attractive therapeutic value in the subversion of the protumor microenvironment and in overcoming chemoimmune resistance.

Levels of p27(kip1) determine Aplidin sensitivity.

Aplidin (plitidepsin) is a novel anticancer drug isolated from the marine tunicate Aplidium albicans. Aplidin shows potent antitumor activity in preclinical models against a wide variety of human tumors. Aplidin is currently in phase II clinical trials in a variety of solid tumors and hematologic malignancies. Moreover, clinical studies of Aplidin in combination with other agents are ongoing because it generally lacks cross-resistance with other known cytotoxic drugs. The mode of action of Aplidin in tumor cells is only partially understood. Aplidin induces an early oxidative stress response, which results in a rapid and sustained activation of the epidermal growth factor receptor, the nonreceptor protein tyrosine kinase Src, and the serine threonine kinases c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase. Here, we show that sensitivity to Aplidin correlates inversely with the levels of expression of the cyclin-dependent kinase inhibitor p27(kip1) (p27) in a panel of low passaged human sarcoma cell lines. Aplidin induces p27 through an oxidation-dependent mechanism and the reduction of p27 levels by specific short hairpin RNA increases Aplidin sensitivity. We confirmed these results in p27 null mouse embryonic fibroblasts corroborating the specificity of the p27 role in Aplidin response because p21(waf1) null mouse embryonic fibroblasts do not show this increased sensitivity. We propose a mechanism of action of Aplidin involving p27 and support the analysis of p27 in the response to Aplidin in currently ongoing clinical trials to establish the levels of this protein as response predictor.

Adding pharmacogenomics to the development of new marine-derived anticancer agents.

Nature has always been a highly productive tool in the development of anticancer therapies. Renewed interest in the potential of this tool has recently been sparked by the realization that the marine ecosystem can be used for the discovery and development of new compounds with clinical potential in advanced resistant tumors. These compounds can be incorporated into combination approaches in a chronic therapy scenario. Our marine anticancer program is using the sea to develop new agents with activity in resistant solid tumors and to identify new cellular targets for therapeutic intervention. This review describes the integration of different pharmacogenomic tools in the development of Yondelis, Aplidin and Kahalalide F, three marine-derived compounds currently in Phase II or III development. Our results are reinforcing the targeted selectivity of these agents and opening the gates for customized therapies in cancer patients in the near future.

Trabectedin as a chemotherapy option for patients with BRCA deficiency.

Trabectedin is a marine-derived product that was originally isolated from the Caribbean sea squirt Ecteinascidia turbinata and the first anticancer marine drug to be approved by the European Union. It is currently used as a single agent for the treatment of patients with soft tissue sarcoma after failure of anthracyclines and ifosfamide, or for those patients who are unsuited to receive these agents, and in patients with relapsed, platinum-sensitive ovarian cancer in combination with pegylated liposomal doxorubicin. Trabectedin has a unique multi-faceted mechanism of action that involves transcription regulation and DNA repair systems, including transcription-coupled nucleotide excision repair and homologous recombination repair (HRR) as the main hallmarks of its antiproliferative activity. In addition, trabectedin has shown the ability to modulate the tumor microenvironment. Indeed, the activity of trabectedin is related to altered function and expression of DNA repair genes, such as BRCA1 (BReast-CAncer susceptibility gene 1) and BRCA2. The particular sensitivity of sarcoma, ovarian and breast cancer cells deficient in HRR, previously observed in preclinical models, now has been confirmed in the clinical setting as well, suggesting that BRCA mutations are associated with improved clinical responses to trabectedin. Current efforts are focused on the evaluation of these unique features of trabectedin and on the identification of predictive factors for patients with an objective to determine whether a deficiency of HRR DNA repair pathway could impact the clinical benefit achieved from trabectedin.

Marine compounds inhibit growth of multiple myeloma in vitro and in vivo.

PURPOSE: The prognosis of patients with multiple myeloma (MM) is still dismal despite recent improvements achieved by introducing new therapeutic agents. However, there remains an urgent need for progress in myeloma drug development. We here show that novel marine-derived compounds can exert potent anti-myeloma activity. EXPERIMENTAL DESIGN: Nine marine-derived compounds were applied at low nM concentrations (0.1-100 nM) to MM cell lines (OPM-2, NCI-H929, U266, RPMI-8226), to primary human myeloma cells and to peripheral blood mononuclear cells. Apoptosis was determined by flow cytometry. In addition, eGFP-transgenic MM cell lines growing with mesenchymal cells from bone marrow were used to visualize tumors by fluorescence stereomicroscopy. Anti-myelomaactivities were studied in vitro in 3D spheroids and in vivo in myeloma xenografts on chicken embryos. Tumor size was analyzed by measuring GFP content with a GFP ELISA. Anti-angiogenic activities of compounds were tested in an in vivo gelatin sponge assay with conditioned media from primary bone marrow-derived endothelial cells. RESULTS: We identified a subset of marine compounds with strong anti-myeloma activity in vitro and in vivo. Moreover, some of the compounds inhibited myeloma-related angiogenesis in the in vivo gelatin sponge assay. They merit further drug development to improve treatment options for MM.

The PARP inhibitor olaparib enhances the sensitivity of Ewing sarcoma to trabectedin.

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.

Epithelial-mesenchymal transition markers and HER3 expression are predictors of elisidepsin treatment response in breast and pancreatic cancer cell lines.

Elisidepsin (elisidepsin trifluoroacetate, Irvalec®, PM02734) is a new synthetic depsipeptide, a result of the PharmaMar Development Program that seeks synthetic products of marine origin-derived compounds. Elisidepsin is a drug with antiproliferative activity in a wide range of tumors. In the present work we studied and characterized the mechanisms associated with sensitivity and resistance to elisidepsin treatment in a broad panel of tumor cell lines from breast and pancreas carcinomas, focusing on different factors involved in epithelial-mesenchymal transition (EMT) and the use of HER family receptors in predicting the in vitro drug response. Interestingly, we observed that the basal protein expression levels of EMT markers show a significant correlation with cell viability in response to elisidepsin treatment in a panel of 12 different breast and pancreatic cancer cell lines. In addition, we generated three elisidepsin treatment-resistant cell lines (MCF-7, HPAC and AsPC-1) and analyzed the pattern of expression of different EMT markers in these cells, confirming that acquired resistance to elisidepsin is associated with a switch to the EMT state. Furthermore, a direct correlation between basal HER3 expression and sensitivity to elisidepsin was observed; moreover, modulation of HER3 expression levels in different cancer cell lines alter their sensitivities to the drug, making them more resistant when HER3 expression is downregulated by a HER3-specific short hairpin RNA and more sensitive when the receptor is overexpressed. These results show that HER3 expression is an important marker of sensitivity to elisidepsin treatment.

Inhibitory effects of marine-derived DNA-binding anti-tumour tetrahydroisoquinolines on the Fanconi anaemia pathway.

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.

Analysis of DNA repair-related genes in breast cancer reveals CUL4A ubiquitin ligase as a novel biomarker of trabectedin response.

Trabectedin is more active in nucleotide excision repair (NER)-efficient and homologous recombination repair (HRR)-deficient cells. As up to 25% of sporadic breast tumors present somatic inactivation of the HRR pathway (BRCAness phenotype), we sought to characterize trabectedin effect in BRCA1-proficient and BRCA1-null breast cancer cell lines. We evaluated whether HRR and NER gene expression correlates with trabectedin sensitivity and explored the response predictive value of the CUL4A ubiquitin ligase, which ubiquitinates NER pathway members. We characterized trabectedin cytotoxicity, cell-cycle effects, and BRCA1, BRCA2, XRCC3, XPG, ERCC1, and CUL4A expression in 10 breast cancer cell lines. Gene expression and trabectedin sensitivity association were determined in cell lines. Survival assays after trabectedin treatment were conducted in CUL4A-silenced BRCA1-proficient and -deficient cells. Because of limited phase II clinical trials evaluating trabectedin efficacy in patients with breast cancer, we assessed CUL4A immunohistochemical staining in a retrospective series of 118 sarcomas from trabectedin-treated patients to validate in vivo our in vitro observations. In cell lines, greater trabectedin sensitivity was associated with higher CUL4A expression and lower BRCA1/ERCC5, BRCA1/CUL4A, and XRCC3/CUL4A expression ratios. In agreement, BRCA1-deficient CUL4A-knockdown cells presented higher cell survival after trabectedin exposure than did scramble control cells. Lack of effect in BRCA1-proficient cells suggests that HRR impairment is key in CUL4A-mediated trabectedin sensitivity. High CUL4A expression in nontranslocation-related patients with sarcoma predicted improved progression-free survival [PFS; HR, 0.37; 95% confidence interval (CI), 0.20-0.68, P = 0.001] and overall survival (OS; HR, 0.44; 95% CI, 0.21-0.93, P = 0.026). Our observations support the notion of greater trabectedin activity in tumors exhibiting BRCAness and reveal CUL4A as a potential biomarker for definition of trabectedin target patients.

Expression of XPG protein in human normal and tumor tissues.

XPG (Xeroderma pigmentosum group G complementing factor) is a protein associated with DNA repair and transcription. Point mutations in ERCC5, the gene coding for XPG, cause the cancer-prone disorder xeroderma pigmentosum (XP) while truncation mutations give rise to individuals with the combined clinical features of XP and Cockayne syndrome. Polymorphisms of ERCC5 or alterations in XPG mRNA expression were also associated to an increase risk of different cancers types and to prognosis of cancer patients. However, the expression of XPG protein in different normal or tumor human tissues is not well known. In the present work, we have validated an immunohistochemistry (IHC) assay for detection of expression levels of XPG protein in FFPE human tissue samples. We have also tested this IHC assay in different normal and tumor human tissues. On a microarray containing 28 normal cores, positive staining was observed in 60% of the samples. The highest staining was detected in adrenal gland, breast, colon, heart, kidney, thyroid and tongue. In tumors, positive staining was observed in 9 of 10 breast cancer samples and in all 5 ovarian cancer and 5 sarcomas samples. Subcellular localization was predominantly nuclear. The use of this validated methodology would help to interpret the role of XPG in tumorogenesis and its use as a possible prognostic or predictive factor.

Plitidepsin (Aplidin) is a potent inhibitor of diffuse large cell and Burkitt lymphoma and is synergistic with rituximab.

Plitidepsin (Aplidin), an antitumor agent of marine origin, presently is undergoing phase II/III clinical trials, and has shown promise for the treatment of lymphoma. Here, we describe the antitumor effects of plitidepsin alone and in combination with rituximab and investigated the effects of each drug and the combination on the cell cycle and mechanism of cell death. Several Diffuse Large Cell Lymphoma (DLCL) lines and Burkitt cell lines were tested for sensitivity to plitidepsin and rituximab. All DLCL and Burkitt lymphoma cell lines were inhibited by plitidepsin in nanomolar concentrations, while rituximab sensitivity varied among different cell lines. Ramos and the RL cell lines proved sensitive to rituximab and were used to test the effects of each of the two drugs. The two agents exhibited synergism at all tested concentrations. For in vivo studies, irradiated athymic nude mice were engrafted with the Ramos lymphoma. Treatment was initiated when the tumors were ~0.5 cm in diameter, and toxic and therapeutic effects were monitored. In the in vivo study, additive effects of the combined two drugs, was demonstrated without an increase in host toxicity. The in vitro synergy and the in vivo additive antitumor effects without an increase in host toxicity with two relatively non-marrow suppressive agents encourages further development of this combination for treatment of aggressive B-cell lymphomas.

ErbB3 expression predicts sensitivity to elisidepsin treatment: in vitro synergism with cisplatin, paclitaxel and gemcitabine in lung, breast and colon cancer cell lines.

Irvalec® (elisidepsin trifluoroacetate, PM02734) is a novel marine-derived cyclic peptide belonging to the Kahaladide family of compounds, currently in clinical trials with preliminary evidence of antitumor activity. Previous studies have shown a correlation between elisidepsin sensitivity and expression of the ErbB3 receptor in a panel of NSCLC cell lines. We have studied the effect of elisidepsin on the ErbB3 pathway, characterizing the expression of all members of the ErbB (HER) family of receptors and their main downstream signaling effectors, such as Akt and MAPK. Interestingly, we observed a downregulation of ErbB3 upon elisidepsin treatment that correlates with a reduction in the Akt phosphorylation levels in the most sensitive cell lines, whereas ErbB3 levels are not affected in the less sensitive ones. Also, we observed that the basal levels of ErbB3 protein expression show a significant correlation with cell viability response against elisidepsin treatment in 14 different cell lines. Furthermore, we analyzed the combination of elisidepsin with different chemotherapeutics agents, such as cisplatin, paclitaxel and gemcitabine, in a panel of different breast (MDA-MB-435, MDA-MB-231 and MCF7), lung (HOP62, DV90 and A549) and colorectal cancer cell lines (DLD1 and HT29). IC50 values for the different drugs were tested. We observed a synergistic effect in all cell lines tested with any chemotherapeutic agent. More importantly, the two in vitro elisidepsin-resistant cell lines (MDA-MB-231 and HOP62) presented a synergistic effect in combination with cisplatin and paclitaxel, respectively. These results provide a rationale for further development of these combinations in an ongoing clinical trial.

PM02734 (elisidepsin) induces caspase-independent cell death associated with features of autophagy, inhibition of the Akt/mTOR signaling pathway, and activation of death-associated protein kinase.

PURPOSE: PM02734 (elisidepsin) is a synthetic marine-derived cyclic peptide of the kahalalide family currently in phase II clinical development. The mechanisms of cell death induced by PM02734 remain unknown. EXPERIMENTAL DESIGN: Human non-small-cell lung cancer (NSCLC) cell lines H322 and A549 were used to evaluate PM02734-induced cytotoxicity, apoptosis, and autophagy, as well as effects on cell death-related signaling pathways. RESULTS: PM02734 at clinically achievable concentrations (0.5-1 µmol/L) was cytotoxic to H322 and A549 cells but did not cause nuclear fragmentation, PARP cleavage, or caspase activation, suggesting that classical apoptosis is not its main mechanism of cell death. In contrast, PM02734-induced cell death was associated with several characteristics of autophagy, including an increase in acidic vesicular organelle content, levels of GFP-LC3-positive puncta, elevation of the levels of Atg-5/12 and LC3-II, and an associated compromise of the autophagic flux resulting in increased number of autophagosomes and/or autolysosomes. Cotreatment with 3-methyladenine (3-MA) and downregulation of Atg-5 gene expression by siRNA partially inhibited PM02734-induced cell death. PM02734 caused inhibition of Akt/mTOR signaling pathways and cotreatment with the Akt inhibitor wortmannin or with the mTOR inhibitor rapamycin led to a significant increase in PM02734-induced cell death. Furthermore, PM02734 caused the activation of death-associated protein kinase (DAPK) by dephosphorylation at Ser308, and downregulation of DAPK expression with siRNA caused also a partial but significant reduction of PM02734-induced cell death. In vivo, PM02734 significantly inhibited subcutaneous A549 tumor growth in nude mice (P < 0.05) in association with induction of autophagy. CONCLUSIONS: Our data indicate that PM02734 causes cell death by a complex mechanism that involves increased autophagosome content, due for the most part to impairment of autophagic flux, inhibition of the Akt/mTOR pathway, and activation of DAPK. This unique mechanism of action justifies the continued development of this agent for the treatment of NSCLC.

Molecular pharmacodynamics of PM02734 (elisidepsin) as single agent and in combination with erlotinib; synergistic activity in human non-small cell lung cancer cell lines and xenograft models.

PM02734 (elisidepsin) is a novel marine-derived cyclic peptide belonging to the Kahalalide family of compounds currently under phase I development with early evidence of a positive therapeutic index. The cytotoxicity of PM02734 has been determined in a panel of human NSCLC (non-small cell lung cancer) cell lines. Western blot analysis showed a direct correlation between ErbB3 expression and cell sensitivity to PM02734. Furthermore, PM02734 was more effective in the induction of ErbB3 degradation and dephosphorylation than in that of ErbB2 and ErbB1 in human NSCLC cell lines. The combination of PM02734 and erlotinib was synergistic in all NSCLC cell lines tested, including erlotinib resistant cell lines, with combination indexes ranging between 0.59 and 0.81. The combination of PM02734 and erlotinib was more effective than either drug alone in mice inoculated intravenously (i.v.) with A549 cells. The combination of PM02734 and erlotinib was more effective in inhibiting AKT than either single agent alone in H322 cells. These results have provided a rational basis for an ongoing clinical trial to explore this combination in patients with advanced malignant solid tumours.