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A critical objective in cancer therapy is to reduce the systemic toxicity through the modification of the biodistribution of anticancer drugs. Herein, we disclose a new biodegradable nanocarrier, polyglutamic acid (PGA) nanocapsules, and present the in vivo pharmacokinetics/toxicity proof-of-concept for the anticancer drug plitidepsin. These novel nanocapsules were prepared using a modified solvent displacement technique where the polyamino acid was electrostatically deposited onto the lipid core. The nanocapsules exhibited an average size of 200 nm, a negative zeta potential and a great capacity for the encapsulation of plitidepsin (encapsulation efficiency above 90%). In addition, the nanocapsules could be freeze-dried and showed an adequate stability profile upon storage. Finally, the in vivo proof-of-concept studies performed in mice indicated that the encapsulation provided the drug with a prolonged blood circulation and a significantly reduced toxicity. In fact, the maximum tolerated dose of the nanoencapsulated drug was more than 3 times that of the reference formulation (Cremophor® EL plitidepsin solution). Overall, beyond the value of this specific formulation, the work reported here represents the evidence of the potential of polyamino acid nanocapsules in nano-oncological therapy The authors would like to acknowledge financial support from CENIT-NANOFAR XS53 project, PharmaMar, Spain, the Ministry of Sciences and Innovation ((CTQ2009-10963), the Xunta de Galicia (Competitive Reference Groups-FEDER funds Ref. 2010/18, and CN2011/037) and the European Commission FP7 EraNet — EuroNanoMed Program-Instituto Carlos III (Lymphotarg proyect, Ref. PS09/02670). Giovanna Lollo has a fellowship from the Ministry of Education of Spain. Marcos Garcia Fuentes acknowledges an Isidro Parga Pondal Fellowship from Xunta de Galicia SI

This is an open access article distributed under the Creative Commons Attribution License.-- et al., Plitidepsin is an antitumor drug of marine origin currently in Phase III clinical trials in multiple myeloma. In cultured cells, plitidepsin induces cell cycle arrest or an acute apoptotic process in which sustained activation of c-Jun N-terminal kinase (JNK) plays a crucial role. With a view to optimizing clinical use of plitidepsin, we have therefore evaluated the possibility of using JNK activation as an in vivo biomarker of response. In this study, we show that administration of a single plitidepsin dose to mice xenografted with human cancer cells does indeed lead to increased phosphorylation of JNK in tumors at 4 to 12 h. By contrast, no changes were found in other in vitro plitidepsin targets such as the levels of phosphorylated-ERK, -p38MAPK or the protein p27KIP1. Interestingly, plitidepsin also increased JNK phosphorylation in spleens from xenografted mice showing similar kinetics to those seen in tumors, thereby suggesting that normal tissues might be useful for predicting drug activity. Furthermore, plitidepsin administration to rats at plasma concentrations comparable to those achievable in patients also increased JNK phosphorylation in peripheral mononuclear blood cells. These findings suggest that changes in JNK activity provide a reliable biomarker for plitidepsin activity and this could be useful for designing clinical trials and maximizing the efficacy of plitidepsin., This work has been partially supported by grants (Programa Cenit, CEN-20091016, SAF2010-18302 and Fondo Europeo de Desarrollo Regional-Instituto de Salud Carlos III, RD12/0036/0021) from Ministerio de Economía y Competitividad of Spain.

Vascular supply of tumors is one of the main targets for cancer therapy. Different studies have shown that most of the microtubule-binding agents present antiangiogenic and vascular-disrupting effects. PM060184 is a new marine-derived drug that binds to a new site in β-tubulin inhibiting tubulin polymerization. The compound is currently being evaluated in phase 1/2 studies in patients with advanced malignancies. The present study describes the antiangiogenic and vascular-disrupting properties of PM060184. Treatment of HUVEC endothelial cells with PM060184 resulted in depolymerization of the microtubule network. Differently from untreated cells, PM060184-treated cells showed a rounded morphology with a well-developed actin filament structure and plasma membrane blebs. Using transwell chambers pre-coated with matrigel™ or collagen matrix, we showed that PM060184 inhibited migration and invasion of HUVEC endothelial cells. We finally found that PM060184 interfered with the correct formation of the HUVEC capillary network grown on matrigel™; moreover, drug treatment also resulted in a significant disruption of the previously formed capillary-like network. This rapid collapse of the endothelial tubular network in vitro occurred in a concentration-dependent manner and was observed at concentrations lower than those affecting cells survival. These in vitro findings were confirmed in nude mice bearing MDA-MB-231 (breast) xenografts: after the administration of a single dose of PM060184 (at its MTD, 16 mg/kg,) a very strong reduction in the number of vessels was quantified in serial tumor sections stained with H&E. Moreover, nude mice xenografted with H-460 (NSCLC) tumors and treated with a single dose of either 2 or 16 mg/kg of PM060184, experienced a strong and dose-dependent decrease of the intratumoral fluorescence after the administration of Angiosense™ 680, a fluorescence vascular imaging probe. Altogether, these data suggest that an anti-vascular mechanism is also contributing to the anti-tumor activities of PM060184. Citation Format: Carlos M. Galmarini, Maria J. Guillen, Juan F. Martinez-Leal, Marta Martinez-Diez, Pablo Aviles. Anti-angiogenic properties of PM060184. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3066.

BACKGROUND: Ewing sarcoma is a bone and soft tissue sarcoma characterized by fusion of the wild-type EWSR1 and FLI1 genes, which creates a constitutively activated transcription factor, EWS/FLI1. Ewing sarcoma cells are dependent on the continued activity of EWS/FLI1 to block differentiation and promote proliferation. For these reasons, EWS/FLI1 represents an ideal drug target. With this in mind, we have shown that the small molecule trabectedin inhibits EWS/FLI1 activity and synergizes with irinotecan in vivo. In addition, we showed that the gene expression response of Ewing sarcoma cells to trabectedin treatment is identical to keratinocytes exposed to UV light. Interestingly, wild-type EWSR1 accumulates in the nucleolus in response to UV light damage. Therefore, we hypothesize that the response and sensitivity of Ewing sarcoma cells to trabectedin is due to the re-localization of EWS/FLI1 in the nucleus. METHODS: Here, we utilize confocal microscopy and chromatin immunoprecipitation to characterize the localization of EWS/FLI1 and EWS/FLI1 deletion mutants in response to trabectedin. Because trabectedin has a narrow therapeutic index, we characterize the effect of PM01183, a second-generation structural analog with an improved pharmacokinetic profile, on EWS/FLI1 localization. In addition, we use qPCR and western blotting to determine the kinetics of EWS/FLI1 inhibition in response to this class of compounds. W demonstrate the effects of PM01183 treatment on the EWS/FLI1 gene signature genome-wide. Finally, we translate these findings in vivo using two Ewing sarcoma xenograft models. RESULTS: Treatment of Ewing sarcoma cells with Trabectedin and PM01183 causes EWS/FLI1 to accumulate in the nucleolus. This effect is accompanied by a loss of binding at target sequences and a reversal in expression of the gene signature of EWS/FLI1. We found the effect on EWS/FLI1 to be sustained, even after removal of the drug from the cell culture medium. We extended these findings in vivo to demonstrate the efficacy of a previously characterized combination therapy. Interestingly, treatment of Ewing sarcoma xenografts with the combination therapy caused the tumor to be replaced by benign adipocytes. CONCLUSIONS: We characterize a novel method of targeting an oncogenic fusion protein by leveraging a property of the wild-type protein to inactivate the oncogenic transcription factor. We demonstrate that wild-type EWSR1 protein's propensity to accumulate in the nucleolus upon UV light damage is maintained within the oncogenic EWS/FLI1 protein with trabectedin or PM01183 treatment. We believe that this accounts for the heightened sensitivity of Ewing sarcoma cells to this class of compounds and has numerous clinical implications. Citation Format: Matt Harlow, Matthew Easton, Maria Jose Guillén Navarro, Lisa Turner, Galen Hostetter, Carlos Galmarini, Pablo Aviles, Patrick Grohar. PM01183 inactivates the EWS/FLI1 transcription factor by redistributing the protein within the nucleus. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-177.

PM050489 is a tubulin-binding agent originally isolated from the marine sponge Lithoplocamia lithistoides. This compound binds with very high affinity to β-tubulin at a new site, disrupting the microtubule network and impairing its function during divison, which leads to mitotic aberrations. MI130004 (a novel antibody-drug conjugate formed by PM050489, a non-hydrolysable linker and trastuzumab) was tested for its in vitro and in vivo activity against selected tumor cell lines with different levels of HER2 expression. In vitro MI130004 showed very high potency and good selectivity for tumor cells that overexpressed HER2, namely HCC-1954 (IC50, 0.036 μg/mL), SK-BR-3 (IC50, 0.017 μg/mL) and BT-474 (IC50, 0.156 μg/mL). At the cellular level, MI130004 impaired tubulin polymerization, causing disorganization and disintegration of the microtubule network which ultimately led to mitotic failure. Expressing HER2 cells of breast (BT-474 and JIMT-1), gastric (Gastric-008 and N87) and ovarian (SK-OV-3 and A2780cis) as well as negative HER2 breast (MDA-MB-231) and gastric (Hs748t) were subcutaneously implanted into immunosuppressed (SCID or athymic) mice. Tumor (ca. 115 mm3) bearing animals (N = 10/group) were randomly allocated to receive the ADC treatments (at different doses) or the appropriate control (placebo included). Treatments were administered weekly for 5 consecutive weeks. Tumor growth (as median/group) was calculated 2-3 times per week. Twenty-four hours after the first dose, representative tumors were dissected free and processed for HER2 expression (erb2) and chromatin organization (Hoescht 33258). The treatment with MI130004 induced a long lasting antitumor effect with statistically significant increases (P Citation Format: Pablo Aviles, Maria Jose Guillen, Juan Manuel Dominguez, Carlos M. Galmarini, Carmen Cuevas. MI130004, a new ADC with a payload of marine origin shows outstanding activity against HER2-expressing tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A147.

This is an open-access paper.-- et al., [Background]: Although the majority of patients with acute myeloid leukemia initially respond to conventional chemotherapy, relapse is still the leading cause of death, probably because of the presence of leukemic stem cells that are insensitive to current therapies. We investigated the antileukemic activity and mechanism of action of zalypsis, a novel alkaloid of marine origin. [Design and Methods]: The activity of zalypsis was studied in four acute myeloid leukemia cell lines and in freshly isolated blasts taken from patients with acute myeloid leukemia before they started therapy. Zalypsis-induced apoptosis of both malignant and normal cells was measured using flow cytometry techniques. Gene expression profiling and western blot studies were performed to assess the mechanism of action of the alkaloid. [Results]: Zalypsis showed a very potent antileukemic activity in all the cell lines tested and potentiated the effect of conventional antileukemic drugs such as cytarabine, fludarabine and daunorubicin. Interestingly, zalypsis showed remarkable ex vivo potency, including activity against the most immature blast cells (CD34+ CD38− Lin−) which include leukemic stem cells. Zalypsis-induced apoptosis was the result of an important deregulation of genes involved in the recognition of double-strand DNA breaks, such as Fanconi anemia genes and BRCA1, but also genes implicated in the repair of double-strand DNA breaks, such as RAD51 and RAD54. These gene findings were confirmed by an increase in several proteins involved in the pathway (pCHK1, pCHK2 and pH2AX). [Conclusions]: The potent and selective antileukemic effect of zalypsis on DNA damage response mechanisms observed in acute myeloid leukemia cell lines and in patients’ samples provides the rationale for the investigation of this compound in clinical trials., This work was supported by a grant from the Ministry of Science and Innovation of Spain (BFU2006-01813/BMC and RD06/0020/ 0041). The CIC receives support from the European Community through the regional development funding program (FEDER). This work was also supported by the ‘Acción Transversal del Cáncer’ project, through an agreement between the Instituto de Salud Carlos III (ISCIII), the Spanish Ministry of Science and Innovation, and the Cancer Research Foundation of Salamanca University. Our group also receives support from the Junta de Castilla y Léon through ‘Ayudas destinadas a financiar programas de actividad investigadora a realizar por grupos de investigación de excelencia de Castilla y León’.

Multiple myeloma (MM) remains incurable, and new drugs with novel mechanisms of action are still needed. In this report, we have analyzed the action of Zalypsis, an alkaloid analogous to certain natural marine compounds, in MM. Zalypsis turned out to be the most potent antimyeloma agent we have tested so far, with IC50 values from picomolar to low nanomolar ranges. It also showed remarkable ex vivo potency in plasma cells from patients and in MM cells in vivo xenografted in mice. Besides the induction of apoptosis and cell cycle arrest, Zalypsis provoked DNA double-strand breaks (DSBs), evidenced by an increase in phospho-histone-H2AX and phospho-CHK2, followed by a striking overexpression of p53 in p53 wild-type cell lines. In addition, in those cell lines in which p53 was mutated, Zalypsis also provoked DSBs and induced cell death, although higher concentrations were required. Immunohistochemical studies in tumors also demonstrated histone-H2AX phosphorylation and p53 overexpression. Gene expression profile studies were concordant with these results, revealing an important deregulation of genes involved in DNA damage response. The potent in vitro and in vivo antimyeloma activity of Zalypsis uncovers the high sensitivity of tumor plasma cells to DSBs and strongly supports the use of this compound in MM patients.

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay was developed and validated to quantify a novel antineoplastic agent, PM02734, in dog plasma. The method was validated to demonstrate the specificity, limit of quantification (LOQ), accuracy, and precision of measurements. The calibration range for PM02734 was established using PM02734 standards from 0.05 to 100 ng/mL in blank plasma. The dominating ions were doubly charged molecular ions [M+2H]2+ at m/z 740.0 instead of singly charged ones at m/z 1478.4. The selected reaction monitoring (SRM), based on the m/z 740.0 --212.2 transition, was specific for PM02734, and that based on the m/z 743.8 --212.2 transition was specific for deuterated PM02734 (the internal standard, IS); no endogenous materials interfered with the analysis of PM02734 and IS from blank plasma. The assay was linear over the concentration range 0.05-100 ng/mL. In terms of sensitivity of assay 0.05 ng/mL is a very low LLOQ, especially considering PM02734 is a peptide. The correlation coefficients for the calibration curves ranged from 0.9990 to 0.9999. The mean intraday and interday accuracies for all calibration standards (n = 9) ranged from 93 to 111% (or =11% bias) in dog plasma, and the mean interday precision for all calibration standards was less than 6.4%. The mean intra- and interday assay accuracy for all quality control replicates in dog plasma (n = 9), determined at each QC level throughout the validated runs, ranged from 85-111% (or =15% bias) and from 99-109% (or =9% bias), respectively. The mean intra- and interday assay precision was less than 12.1 and 13.3% for all QC levels, respectively. The assay has been used to support preclinical pharmacokinetic (PK) and toxicokinetic studies. The results showed that preclinical samples could be monitored for PM02734 up to 168 h after dosing, which allowed us to identify multiple elimination phases and accurately estimate PK information.

PM01218 is a novel marine-derived alkaloid and has shown potent growth inhibitory activity against several human cancer cell lines. A rapid and sensitive high performance liquid chromatography/tandem mass spectrometry (HPLC–MS/MS) method was developed and validated to quantify PM01218 in mouse and rat plasma. The lower limit of quantitation (LLOQ) was 0.05 ng/mL. The calibration curve was linear from 0.05 to 100 ng/mL ( R 2 > 0.999). The assay was specifically based on the multiple reaction monitoring (MRM) transitions at m / z 278.4 → 184.2, no endogenous material interfaced with the analysis of PM01218 and its internal standard from blank mouse and rat plasma. The mean intra- and inter-day assay accuracy remained below 15 and 8%, respectively, for all calibration standards and QC samples. The intra- and inter-day assay precision was less than 12.8 and 8.5% for all QC levels, respectively. The utility of the assay was demonstrated by pharmacokinetics studies of i.v. (bolus) PM01218 on SD rats.

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay was developed and validated to quantify a novel antineoplastic agent, PM00104, in mouse, rat, dog, and human plasma. The method was validated to demonstrate the specificity, limit of quantification (LOQ), accuracy, and precision of measurements. The calibration range for PM00104 was established using PM00104 standards from 0.01-5.0 ng/mL in blank plasma. The selected reaction monitoring (SRM), based on the m/z 692.2 --> 218.2 transition, was specific for PM00104, and that based on the m/z 697.2 --> 218.2 transition was specific for PM00104 ((13)C(2),(2)H(3)) (the internal standard, IS); no endogenous materials interfered with the analysis of PM00104 and IS from blank plasma. The assay was linear over the concentration range 0.01-5.0 ng/mL. The correlation coefficients for the calibration curves ranged from 0.9981-0.9999. The mean intra-day and inter-day accuracies for all calibration standards (n = 8) ranged from 97-105% (< or =5% bias) in human plasma, and the mean inter-day precision for all calibration standards was less than 8.5%. The mean intra- and inter-day assay accuracy for all quality control (QC) replicates in human plasma (n = 9), determined at each QC level throughout the validated runs, ranged from 96-112% (< or =12% bias) and from 102-105% (< or =5% bias), respectively. The mean intra- and inter-day assay precision was less than 15.0 and 11.8% for all QC levels, respectively. For the QC samples prepared in animal species plasma, the %CV values of the assays ranged from 1.8-8.8% in mouse plasma, from 3.7-13.8% in rat plasma, and from 3.0-7.2% in dog plasma. The assay accuracies ranged from 92-102% (< or =8% bias) for all QC levels prepared in mouse plasma; ranged from 93-106% (< or =7% bias) in rat plasma; and ranged from 95-114% (< or =14% bias) in dog plasma. The assay has been used to support preclinical pharmacokinetic and toxicokinetic studies and is currently used to measure PM00104 plasma concentrations to support clinical trials.

A sensitive high-performance liquid chromatography-tandem mass spectrometry assay for thiocoraline, an anti-tumor depsipeptide, in mouse plasma is described. Echinomycin, a quinoxaline peptide, was used as an internal standard. Thiocoraline was recovered from the mouse plasma using protein precipitation with acetonitrile and followed by solid-phase extraction of the supernatant. The mobile phase consisted of methanol (0.1% formic acid)-water (0.1% formic acid) (90:10, v/v). The analytical column was a YMC C(18). The standard curve was linear from 0.1 to 50 ng/ml (R(2)>0.99). The lower limit of quantitation was 0.1 ng/ml. The assay was specific based on the multiple reaction monitoring transitions at m/z 1157-->215 and m/z 1101-->243 for thiocoraline and the internal standard, echinomycin, respectively. The mean intra- and inter-day assay accuracies remained below 5 and 12%, respectively, for all calibration standards and quality control (QC) samples. The intra- and inter-day assay precisions were less than 11.4 and 9.5% for all QC levels, respectively. The utility of the assay was demonstrated by a pharmacokinetic study of i.v. (bolus) thiocoraline on CD-1 mice. Thiocoraline was stable in mouse plasma in an ice-water bath for 6 h and for three freeze-thaw cycles. The reconstituted thiocoraline after extraction and drying sample process was stable in the autosampler for over 24 h. The assay was able to quantify thiocoraline in plasma up to 48 h following dose. Pharmacokinetic analysis showed that thiocoraline has distinct pharmacokinetic profiling when dosed in different formulation solutions. The assay is currently used to measure thiocoraline plasma concentrations in support of a project to develop a suitable formulation with a desirable pharmacokinetic profile.

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay was developed and validated to quantify a novel marine-derived depsipeptide, Aplidin, in human plasma. The method was validated to demonstrate the specificity, recovery, limit of quantitation (LOQ), accuracy, and precision of measurements. The calibration range for Aplidin was established using Aplidin standards from 0.05-50 ng/mL in blank human plasma. The multiple reaction monitoring, based on the transition m/z 1110.7 --295.3, was specific for Aplidin, and that based on the transition m/z 1112.6 --297.3 was specific for didemnin B (the internal standard); no endogenous materials interfered with the analysis of Aplidin and didemnin B from blank human plasma. The assay was linear over the concentration range 0.05-50.0 ng/mL. The correlation coefficients for the calibration curves ranged from 0.9979 to 0.9999. The mean intra- and interday accuracies for all calibration standards (n = 12) ranged from 97 to 106% (/=6% bias), and the mean interday precision for all calibration standards was less than 8.3%. The mean intra- and interday assay accuracy for all quality control replicates (n = 12), determined at each QC level throughout the validated runs, remained below 12 and 7%, respectively. The mean intra- and interday assay precision was less than 13.1 and 10.7% for all QC levels, respectively. The assay is currently used to measure Aplidin plasma concentrations to support clinical trials.

breast and platinum-resistant/refractory ovarian) clinical evaluation. In vitro studies demonstrate that lurbinectedin causes delayed progression of S phase and cycle arrest in G2M. Also, lurbinectedin was 3−4 time less potent in NER deficient cells whereas it was 100 time more potent in HR deficient cells. The in vivo antitumor activity of lurbinectedin was evaluated in human ovarian carcinoma models, growing subcutaneously (OCX) or in the peritoenal cavity (IPX). Material and Methods: The in vivo pharmacological behavior of lurbinectedin was evaluated in M5076 (murine reticulosarcoma), MN-MCA1 (murine fibrosarcoma), 2 human OCX (HOC 18 and MNB-PTX-1) and 2 IPX (HOC8 and HOC22). Lurbinectedin was given i.v. at 0.2 mg/kg/day on days 0, 7, 14 (q7d×3). The antitumor effect was calculated either by T/C (as a percentage of the change in tumor size for treated [T] and placebo [C]) or by the increase of like span (ILS) (as 100·[(T − C)/C], where ‘T’ and ‘C’ are the median survival time of the treated and placebo); treatment-related anti-metastic activity was also evaluated. Results: In M5076 lurbinectedin was only marginally active (T/C, 73.4%) against the primary tumor whereas in MN-MCA1 demonstrated a good antitumor activity (best T/C 15%). In both tumor models lurbinectedin produced a striking antimetastatic effect (p < 0.001). Lurbinectedin treatment resulted in no antitumor activity in the MNB-PTX-1 model. In animals bearing OCX HOC18 tumors, lurbinectedin treatment induced a strong and long-lasting antitumor effect (minimal T/C, 13% Day 16). Tumor volume were reduced (P < 0.01) compared to placebo on days 10−17. Also, lurbinectedin treatment reduced tumor growth rate: all animals were sacrificed due to tumor volume on Day 63, later than placebo (Day 15). Lurbinectedin treatment of HOC8 IPX xenografts resulted in (P < 0.05) antitumor activity compared to untreated animals (ILS % of 140.0). Eight out of 9 of mice bearing HOC22 IPX tumors and treated with lurbinectedin survived 4 months after the death of the last placebo-treated animal and, then considered as tumor-free. Conclusions: The in vivo antitumor effect of lurbinectedin was demonstrated in a panel of human and murine experimental models, characterized by different behaviors and drug sensitivity.

[Background] Zalypsis® is a marine compound in phase II clinical trials for multiple myeloma, cervical and endometrial cancer, and Ewing’s sarcoma. However, the determinants of the response to Zalypsis are not well known. The identification of biomarkers for Zalypsis activity would also contribute to broaden the spectrum of tumors by selecting those patients more likely to respond to this therapy., [Methods] Using in vitro drug sensitivity data coupled with a set of molecular data from a panel of sarcoma cell lines, we developed molecular signatures that predict sensitivity to Zalypsis. We verified these results in culture and in vivo xenograft studies., [Results] Zalypsis resistance was dependent on the expression levels of PDGFRα or constitutive phosphorylation of c-Kit, indicating that the activation of tyrosine kinase receptors (TKRs) may determine resistance to Zalypsis. To validate our observation, we measured the levels of total and active (phosphorylated) forms of the RTKs PDGFRα/β, c-Kit, and EGFR in a new panel of diverse solid tumor cell lines and found that the IC50 to the drug correlated with RTK activation in this new panel. We further tested our predictions about Zalypsis determinants for response in vivo in xenograft models. All cells lines expressing low levels of RTK signaling were sensitive to Zalypsis in vivo, whereas all cell lines except two with high levels of RTK signaling were resistant to the drug., [Conclusions] RTK activation might provide important signals to overcome the cytotoxicity of Zalypsis and should be taken into consideration in current and future clinical trials., The AC lab was supported by grants to from the Spanish Ministry of Economy and Competitivity, ISCIII (Fis: PI12/00137, RTICC: RD12/0036/0028), Consejeria de Ciencia e Innovacion (CTS-6844), and Consejeria de Salud of the Junta de Andalucia (PI-0135-2010 and PI-0306-2012). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Pneumocystis carinii is an important opportunistic pathogen that remains a significant cause of lethal pneumonia in immunocompromised individuals such as patients with AIDS and patients receiving chemotherapy or immunosuppressive drugs for organ transplantation or other pathological conditions. Patients suffering from P. carinii pneumonia (PCP) are usually treated with trimethoprim-sulfamethoxazole (TMP-SMX) or pentamidine (24). However, the relatively high frequency of adverse reactions to these drugs reflects the need for new therapeutic approaches. For this reason, the pharmaceutical industry is investigating more effective and less toxic agents. Sordarin derivatives are a new class of antifungal agents that target protein synthesis (11), with marked in vitro activity against P. carinii (13) and excellent in vivo activity in experimental PCP (E. Dei-Cas, E. M. Aliouat, C. Mullet, E. Mazars, and D. Gargallo, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F-65, 1997). Well-defined mouse, rat, or rabbit experimental models (2, 4, 22) can be used to describe the in vivo anti-PCP activity of new compounds. Several in vitro tests for evaluating compound activity against P. carinii have been described using axenic cultures or coculture with feeder cells (8, 10, 14). However, no universally accepted standard method is presently available for the in vitro evaluation of anti-P. carinii molecules (10). The anti-Pneumocystis activity of any given antimicrobial could be evaluated in terms of its intrinsic activity (in vitro) and serum time profile (in vivo) (12). However, the results obtained with different in vitro assays (8, 10) yield limited information on the intrinsic activity of anti-Pneumocystis compounds. Furthermore, comparisons between product activities and extrapolation to in vivo activity remain unreliable. The Hill equation, which describes sigmoid concentration-effect relationships, has proven its utility by revealing in vitro pharmacodynamic properties of several antibiotics (17, 30). This approach offers at least three parameters which can be used to describe the in vitro activity of antimicrobial compounds (17): the maximum effect (Emax) as a measure of efficacy, the 50% effective concentration (EC50) as a parameter of intrinsic activity, and the slope (γ) of the concentration-effect relationship. The aim of the present work was to establish the experimental conditions allowing definition of the in vitro pharmacodynamic parameters of tested drugs for defining the intrinsic activity of anti-Pneumocystis molecules, as well as the relationships between their in vitro activities and in vivo effects on microorganisms. (This work was presented in part at the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, Calif., 24 to 27 September, 1998 [E. M. Aliouat, P. Aviles, E. Dei-Cas, E. Herreros, L. Dujardin, and D. Gargallo-Viola, abstr. J-15].)

Background: Lurbinectedin (PM01183), a new synthetic tetrahydroisoquinoline alkaloid, is a minor groove specific DNA binder that interacts directly with specific factors involved in DNA repair and transcription pathways. In living cells, PM01183-DNA adducts stall replication and transcription giving rise to double strand breaks, inducing accumulation of cells in the S-phase of the cell cycle and triggering apoptosis. In both the ongoing phase II trials (pancreas and platinum-resistant ovarian cancer) the first stage was concluded with positive results in clinical activity, ensuring the continuity of the trials. Also, phase I trials in combination with doxorubicin and gemcitabine as well as in advanced acute leukemia are ongoing. The in vitro and in vivo synergism of PM01183+ topoisomerase I inhibitors is presented here. Material and Methods: In vitro: PM01183+topotecan synergism was tested in a panel of 12 tumor cell lines. In vivo: Athymic mice were implanted with SW1990 (pancreas), HT29 (colon) or H460 (NSCLC) cancer cells. Mice bearing HT29 or H460 tumors (ca. 150 mm3) were allocated to 13 groups (N=6-8/group): placebo; PM01183 at MTD (0.180 mg/kg [0.54 mg/m2]), 0.75MTD, 0.5MTD and 0.25MTD; irinotecan at MTD (50 mg/kg [150 mg/m2]), 0.75MTD, 0.5MTD and 0.25MTD; and, PM01183+irinotecan at (1+1), (0.75+0.75), (0.50+0.50) and (0.25+0.25) MTDs. Animals bearing SW1990 tumors received treatments (single or combination) at the MTD levels. Treatments were given intravenously once per week (PM01183) or every 4 days (irinotecan) during the placebo-treated survival time. The combination index (CI) was determined by the CI-isobol method. Results: The combination of PM01183 plus topotecan in NSCLC (A549), melanoma (SK-MEL-2), hepatocarcinoma (HepG2), colon (HT29), pancreas (PANC-1) and glioma (U87MG) cells was considered synergistic. In vivo, a synergistic antitumor effect (CI Conclusion: PM01183 synergizes the in vitro effect of topotecan in several tumor cell lines including NSCLC, melanoma, hepatocarcinoma, colon, pancreas and glioma. In agreement with the in vitro results, the treatment of mice xenografted with colon, NSCLC or pancreas tumors with PM01183 combined with a topoisomerase I inhibitor (irinotecan) resulted in synergistic antitumor activity. Citation Format: Carlos Galmarini, Maria José Guillen, Victoria Moneo, Luis Francisco Garcia, Mandy Palomares, Oscar Cataluña, Carmen Cuevas, Pablo Avilés. Lurbinectedin (PM01183) synergizes with topoisomerase I inhibitors in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5499. doi:10.1158/1538-7445.AM2013-5499

Background: Lurbinectedin (PM01183), a new synthetic tetrahydroisoquinoline alkaloid, is a minor groove specific DNA binder that interacts directly with specific factors involved in DNA repair and transcription pathways. In living cells, PM01183-DNA adducts stall replication and transcription giving rise to double strand breaks, inducing accumulation of cells in the S-phase of the cell cycle and triggering apoptosis. In both the ongoing phase II trials (pancreas and platinum-resistant ovarian cancer) the first stage was concluded with positive results in clinical activity, ensuring the continuity of the trials. Also, phase I trials in combination with doxorubicin and gemcitabine as well as in advanced acute leukemia are ongoing. In vivo studies aimed at defining the synergism of PM01183 combined with taxanes (taxol and docetaxel) are presented here. Material and Methods: Athymic mice were implanted with HGC-27 (gastric), A2780 (ovarian), H460 (NSCLC), 22RV1 (prostate) or MDA-MB-231 (breast) cancer cells. Mice bearing tumors (ca. 150 mm3) were allocated (N=6-8/group) to experimental groups, namely: placebo; PM01183 at MTD (0.180 mg/kg [0.54 mg/m2]), 0.75MTD, 0.5MTD and 0.25MTD; taxol (H460, A2780 or HGC-27) or docetaxel (22RV1 or MDA-MB-231) at their MTD, 0.75MTD, 0.5MTD and 0.25MTD; and, PM01183 combined either with taxol or docetaxel at (1+1), (0.75+0.75), (0.50+0.50) and (0.25+0.25) MTDs. All treatments were given intravenously once per week during the placebo-treated survival time. The combination index (CI) was determined by the CI-isobol method. When needed, statistical differences for the antitumor effect recorded between groups were determined using two-tailed Mann-Whitney U test. Results: In all the experiments, the combination of PM01183 and taxol or docetaxel resulted in more antitumor effect (P Citation Format: Maria José Guillén, Oscar Cataluña, Mandy Palomares, Raquel López, Práxedes Núñez, Carmen Cuevas, Pablo Avilés. Lurbinectedin (PM01183) in vivo synergizes the antitumor activity of taxanes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5495. doi:10.1158/1538-7445.AM2013-5495

Background: Lurbinectedin (PM01183), a new synthetic tetrahydroisoquinoline alkaloid, is a minor groove specific DNA binder that interacts directly with specific factors involved in DNA repair and transcription pathways. In living cells, PM01183-DNA adducts stall replication and transcription giving rise to double strand breaks, inducing accumulation of cells in the S-phase of the cell cycle and triggering apoptosis. In both the ongoing phase II trials (pancreas and platinum-resistant ovarian cancer) the first stage was concluded with positive results in clinical activity, ensuring the continuity of the trials. Also, phase I trials in combination with doxorubicin and gemcitabine as well as in advanced acute leukemia are ongoing. The synergism (in vitro and in vivo) of PM01183 combined with 5-Fluorouracil (5-FU) is presented here. Material and Methods: In vitro: The combination of PM01183+5-FU was tested in 12 different tumor cell lines: lung, sarcoma, melanoma, prostate, pancreas, gastric, ovarian, hepatocarcinoma, colon, kidney, glioma and breast cancer. In vivo: Athymic mice were implanted with HGC-27 (gastric) cancer cells. Mice bearing tumors (ca. 150 mm3) were allocated to 13 groups (N=6-8/group): placebo; PM01183 at MTD (0.180 mg/kg [0.54 mg/m2]), 0.75MTD, 0.5MTD and 0.25MTD; 5-FU at MTD (50.0 mg/kg), 0.75MTD, 0.5MTD and 0.25MTD; and, PM01183+5-FU at (1+1), (0.75+0.75), (0.50+0.50) and (0.25+0.25) MTDs. In separate experiments, mice bearing SW1990 (pancreas) or HT-29 (colon) tumors received treatments (single or combination) at the MTD levels. All treatments were given intravenously once per week during the placebo-treated survival time. The combination index (CI) was determined by the CI-isobol method in the HGC-27 experiments (in vitro or in vivo). Statistical differences for the antitumor effect recorded between groups were determined using two-tailed Mann-Whitney U test. Results: In vitro, a synergistic effect was seen in gastric (HGC-27) and colon (HT29) cells. The recorded in vivo antitumor effect indicated synergism (CI Conclusion: In vitro, the combination of PM01183+5-FU showed a synergistic effect in gastric and colon tumor cell lines. In agreement, in vivo synergism was seen after the treatment with PM01183+5-FU of mice bearing gastric, colon or pancreas xenografted tumors. Citation Format: Pablo Aviles, Maria José Guillen, Carlos Galmarini, Luis Francisco García, Raquel López, Práxedes Núñez, Victoria Moneo, Carmen Cuevas. Synergism of lurbinectedin (PM01183) combined with 5-fluorouracil (5-FU): in vitro and in vivo studies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5498. doi:10.1158/1538-7445.AM2013-5498

breast and platinum-resistant/refractory ovarian) clinical evaluation. In vitro studies demonstrate that lurbinectedin causes delayed progression of S phase and cycle arrest in G2M. Also, lurbinectedin was 3−4 time less potent in NER deficient cells whereas it was 100 time more potent in HR deficient cells. The in vivo antitumor activity of lurbinectedin was evaluated in human ovarian carcinoma models, growing subcutaneously (OCX) or in the peritoenal cavity (IPX). Material and Methods: The in vivo pharmacological behavior of lurbinectedin was evaluated in M5076 (murine reticulosarcoma), MN-MCA1 (murine fibrosarcoma), 2 human OCX (HOC 18 and MNB-PTX-1) and 2 IPX (HOC8 and HOC22). Lurbinectedin was given i.v. at 0.2 mg/kg/day on days 0, 7, 14 (q7d×3). The antitumor effect was calculated either by T/C (as a percentage of the change in tumor size for treated [T] and placebo [C]) or by the increase of like span (ILS) (as 100·[(T − C)/C], where ‘T’ and ‘C’ are the median survival time of the treated and placebo); treatment-related anti-metastic activity was also evaluated. Results: In M5076 lurbinectedin was only marginally active (T/C, 73.4%) against the primary tumor whereas in MN-MCA1 demonstrated a good antitumor activity (best T/C 15%). In both tumor models lurbinectedin produced a striking antimetastatic effect (p < 0.001). Lurbinectedin treatment resulted in no antitumor activity in the MNB-PTX-1 model. In animals bearing OCX HOC18 tumors, lurbinectedin treatment induced a strong and long-lasting antitumor effect (minimal T/C, 13% Day 16). Tumor volume were reduced (P < 0.01) compared to placebo on days 10−17. Also, lurbinectedin treatment reduced tumor growth rate: all animals were sacrificed due to tumor volume on Day 63, later than placebo (Day 15). Lurbinectedin treatment of HOC8 IPX xenografts resulted in (P < 0.05) antitumor activity compared to untreated animals (ILS % of 140.0). Eight out of 9 of mice bearing HOC22 IPX tumors and treated with lurbinectedin survived 4 months after the death of the last placebo-treated animal and, then considered as tumor-free. Conclusions: The in vivo antitumor effect of lurbinectedin was demonstrated in a panel of human and murine experimental models, characterized by different behaviors and drug sensitivity.

breast and platinum-resistant/refractory ovarian) clinical evaluation. In vitro studies demonstrate that lurbinectedin causes delayed progression of S phase and cycle arrest in G2M. Also, lurbinectedin was 3−4 time less potent in NER deficient cells whereas it was 100 time more potent in HR deficient cells. The in vivo antitumor activity of lurbinectedin was evaluated in human ovarian carcinoma models, growing subcutaneously (OCX) or in the peritoenal cavity (IPX). Material and Methods: The in vivo pharmacological behavior of lurbinectedin was evaluated in M5076 (murine reticulosarcoma), MN-MCA1 (murine fibrosarcoma), 2 human OCX (HOC 18 and MNB-PTX-1) and 2 IPX (HOC8 and HOC22). Lurbinectedin was given i.v. at 0.2 mg/kg/day on days 0, 7, 14 (q7d×3). The antitumor effect was calculated either by T/C (as a percentage of the change in tumor size for treated [T] and placebo [C]) or by the increase of like span (ILS) (as 100·[(T − C)/C], where ‘T’ and ‘C’ are the median survival time of the treated and placebo); treatment-related anti-metastic activity was also evaluated. Results: In M5076 lurbinectedin was only marginally active (T/C, 73.4%) against the primary tumor whereas in MN-MCA1 demonstrated a good antitumor activity (best T/C 15%). In both tumor models lurbinectedin produced a striking antimetastatic effect (p < 0.001). Lurbinectedin treatment resulted in no antitumor activity in the MNB-PTX-1 model. In animals bearing OCX HOC18 tumors, lurbinectedin treatment induced a strong and long-lasting antitumor effect (minimal T/C, 13% Day 16). Tumor volume were reduced (P < 0.01) compared to placebo on days 10−17. Also, lurbinectedin treatment reduced tumor growth rate: all animals were sacrificed due to tumor volume on Day 63, later than placebo (Day 15). Lurbinectedin treatment of HOC8 IPX xenografts resulted in (P < 0.05) antitumor activity compared to untreated animals (ILS % of 140.0). Eight out of 9 of mice bearing HOC22 IPX tumors and treated with lurbinectedin survived 4 months after the death of the last placebo-treated animal and, then considered as tumor-free. Conclusions: The in vivo antitumor effect of lurbinectedin was demonstrated in a panel of human and murine experimental models, characterized by different behaviors and drug sensitivity.

Background: Pancreatic ductal adenocarcinoma is the fourth cause of death in the Western world and, despite the advances in cancer therapies, limited therapeutic benefit (palliating symptoms, better quality of life) may be offered to patients: the overall 5-year survival rate remains < 5%. PM01183 is a new synthetic tetrahydroisoquinoline alkaloid that binds to selected DNA sequences and promotes apoptosis by inducing double-strand breaks at nanomolar concentrations. Confirmed antitumor clinical activity was observed at the recommended dose level of PM01183 in the first-in-human Phase I study in patients with advanced/refractory solid tumors. Currently, PM01183 clinical development includes Phase II single agent trials (pancreas, breast and platinum-resistant/refractory ovarian) as well as Phase I trials in combination (with doxorubicin and gemcitabine) and in advanced acute leukemia. The in vivo antitumor activity of PM01183 was investigated in the human NP9 pancreatic orthotopic model Material and Methods: Athymic nude female mice were orthotopically implanted with luciferase-expressing NP9Luc tumors. Fifteen days after implantation, tumor bearing mice were randomly allocated (N=12 /group) into either PM01183 (at 0.18 mg/kg), gemcitabine (at 180 mg/kg) or control (placebo) groups. Treatments were initiated (Day 0) and administered for 3 consecutive weeks (q7dx3; days 0, 7, 14). Response to treatment was assessed on Day 7 (short-term) by bioluminescence imaging (BLI), and by survival evaluation (long-term): mice were sacrificed when their tumor diameters reached ca. 2 cm or when they became moribund. Statistical differences between groups were determined using two-tailed Mann-Whitney U test and by analysis of the Kaplan-Meier curve. Results: Results showed that PM01183 (q7dx3 at 0.18 mg/kg) induced statistically significant tumor reduction compared to placebo-treated animals by BLI analysis (P= 0.006) on Day 7. Reductions in tumor size were associated to changes in survival median times: PM01183 treatment significantly increased the survival time compared to either gemcitabine- (P= 0.003) or placebo-treated (P= 0.0001) animals. Conclusion: PM01183 demonstrated in vivo antitumor activity in a human pancreatic orthotopic model (NP9), as reflected by metabolic, size-related and survival rate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1777. doi:1538-7445.AM2012-1777

Abstract 3003 During the last years, several novel drugs combinations have changed the outcome of Multiple Myeloma (MM) patients; nevertheless, relapse is still the rule for most of these patients, and, therefore, other drugs with novel mechanisms of action are required. The current research in this topic is usually based on the addition of novel drugs to standard of care combinations in order to try to increase their efficacy. Bortezomib + steroids has turned out to be one of these standards, both for induction therapy before high dose melphalan and also for non-trasplant candidates. Zalypsis is a novel marine compound with remarkable preclinical activity in MM, which is currently being explored in a phase I/II trial. We explored the preclinical efficacy of the triple combination of zalypsis, bortezomib and dexamethasone in different models and investigated its mechanism of action. This combination showed high potency when compared to the respective single agents and double combinations, and, in fact, the analysis by the Calcusyn software, evidenced combination indexes in the highly synergistic range (CI 0.2–0.6). This effect was observed not only in the MM1S cell line, but also in freshly isolated cells from MM patients and, most importantly, in a subcutaneous xenograft of a human plasmocytoma in SCID mice. In this last model, we used suboptimal doses of bortezomib and dexamethasone (0.1 mg/Kg and 1 mg/Kg respectively ip, 5 days per week indefinitely) combined with an active dose of zalypsis (0,75 mg/Kg iv weekly for three total doses). Probably due to the low doses used, the double combination of bortezomib + dexamethasone did not show much effect as compared to the control group, and neither bortezomib nor dexamethasone alone could significantly improve the efficacy of Zalypsis. Nevertheless, when the three drugs were combined together, the tumor volume drastically shrunk with differences highly statistically significant (p These results impelled us to investigate the mechanisms relying under the synergy observed. The triple combination induced caspase dependent apoptosis, demonstrated by Annexin V induction and cleavage of caspases 3, 7, 8, 9 and also PARP. By contrast, the activity on the cell cycle was not evident for this combination, indicating a preferential apoptotic effect and not a substantial antiproliferative one. Of note, two mechanisms seem to be specifically associated to the combination of the three agents: 1. A significant release of Endonuclease-G and Cytochrome-C from the mitochondria into the cytosol was observed, suggesting a role for the intrinsic pathway of apoptosis; and 2. One of the most significant changes induced by the combination was an increase in the levels of AIF (apoptosis inducing factor) in the cytosol and nucleus, which is probably indicating the involvement of caspase independent mechanisms. In line with these results, pretreatment with ZVAD was only able to partially rescue from apoptosis, also indicating the potential activation of a dual mechanism: dependent and independent of caspases. Finally, we had previously shown that Zalypsis acts, at least partially, through the induction of DNA damage, based on a p53 dependent mechanism. In this study, the addition of bortezomib and dexamethasone, further increased the H2AX phosphorylation induced by Zalypsis, suggesting a potentiation of the DNA damaging effect; and the combination was also associated with a clear increase in the levels of p53 protein by WB as compared with the agents in monotherapy. The study of the mechanism of action is currently being completed and final results will be presented at the meeting. In summary, the combination of zalypsis + bortezomib + dexamethasone shows high synergy in MM through the activation of different pathways, and these results provide the rationale for its use in relapsed/refractory MM patients. Disclosures: Cuevas: Pharmamar: Employment. Avilés:Pharmamar: Employment. Pandiella:Pharmamar: Research Funding. San Miguel:Pharmamar: Research Funding; Millenium: Consultancy, Research Funding.

This work was focused on the identification of predictive biomarkers for response to Zalypsis®, a bis-tetrahydroisoquinoline derivative structurally related to the marine-derived compounds jorumycin and renieramycins. Zalypsis® binds to the minor groove of DNA through its reactive carbinolamine group, establishing covalent bonds with the amino group of selected guanines in the DNA, with preference for the triplets AGG, GGC, AGC, CGG and TGG. This particular molecular interaction of Zalypsis® with the DNA creates the equivalent to a functional interstrand crosslink that lead to a strong inhibition of the early phases of transcription. Zalypsis® showed potent antitumor activity both in vitro and in vivo, in preclinical models, against a wide variety of human tumors and is currently in phase II clinical trials for solid tumors, showing a tolerable toxicologic profile. In order to gain knowledge on the molecular basis of sensitivity/resistance to Zalypsis®, we used a panel of cell lines from untreated sarcoma tumor samples, keeping the cell lines low passaged to avoid extensive genetic alterations as a consequence of genetic instability of the tumor. These cell lines were extensively characterized for molecular markers and for response to chemotherapeutic drugs, including Zalypsis®. When analyzing the correlation of the sensitivity of these sarcoma cell lines to Zalypsis® and the expression of different molecular markers, it was found that, in general, high basal protein expression levels of PDGFR-a were associated with increased resistance to the drug in vitro. However, among the 20 cell lines studied, two of them expressed low levels of PDGFR-a and were quite resistant to the drug. After analysing other markers, it was found that these cell lines expressed constitutively phosphorylated c-kit receptor, which could have accounted for the observed resistance. To validate the predictive value of PDGFR-a in vivo, and due to the fact that the established sarcoma cells were not tumorigenic in mice, we selected a panel of 9 human epithelial tumor cell lines and tested their sensitivity to Zalypsis® in xenograft models, and, in parallel, the expression of PDGFR-a. HGC27, SW1990, Calu-6 and A2780 showed relative high levels of expression of PDGFR-a in vitro, while UM-UC-3, MX1, MDA-MB-231 and HepG2 showed relative low levels. The subset of cells expressing high levels of PDGFR-a was consistently resistant to Zalypsis®. SKOV-3 cells, which expressed low levels of the marker, were also resistant to the drug. However, after analyzing the expression of other molecular markers, we found that these cells expressed high levels of constitutively phosphorylated EGF receptor that, again, could have accounted for the relative resistance to the drug. Altogether, these results suggest that PDGFR-a could be considered as a useful predictive biomarker of response to Zalypsis®. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1685.

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

Abstract 3792 Poster Board III-728 Background p53 mutation is the most frequent single genetic abnormality found in therapy related AML and is also quite frequent in de novo AML with an incidence between 10% and 25%; Moreover patients with p53 mutations characteristically present complex karyotypes and complicated chromosome rearrangements, leading to an adverse prognosis and resistance to conventional chemotherapeutic agents. Consequently, there is a need for novel antileukemic drugs that could work on both p53-wild type and p53-mutated AML patients. In this study we have evaluated the activity of Zalypsis, a novel alkaloid from marine origin, in several AML cell lines and patients samples with different p53 status. Methods The efficacy of Zalypsis was analyzed in four AML cell lines (HL60, HEL, MV4.11 and KG1) and in cells from fresh bone marrow samples from ten newly diagnosed AML patients. The cytotoxicity was analyzed by means of MTT assay and with a multiparametric flow cytometry (MFC) technique that allowed us to study the efficacy of the drug in both mature and immature blast cell compartments. Proteomic and genomic changes induced after treatment with Zalypsis were analyzed in two AML cell lines with different p53 status (HEL and HL60) by Western-Blot and gene expression profile studies. Results Zalypsis showed a very potent antileukemic activity in the four AML cell lines tested, with IC50s at 48 hours below 1 nM. When compared with the in vitro activity of conventional antileukemic agents, such as cytarabine, doxorubicine or fludarabine, Zalypsis turned out to be 10-100 times more potent. It also showed remarkable ex vivo potency in freshly isolated blasts from ten AML patients. In these patients samples, we had the opportunity to separately analyze by multiparametric flow cytometry, the activity of Zalypsis in the different blasts populations, and we constantly observed similar activity in the most mature blast population and in the most immature one (CD34+, CD38- Lineage-), which is thought to include the leukemic stem cells and which is usually more resistant to conventional chemotherapy. Regarding toxicity, Zalypsis preserved the CD34+ non tumoral hematopoietic progenitor cells. The combination of this novel drug with conventional antileukemic agents indicated that Zalypsis is a good partner for combination with all of them, being the combination with cytarabine and daunorubicin particularly attractive for the clinic. Interestingly, as already said, all cell lines were extremely sensitive to Zalypsis, independently of the p53 status, as some of them displayed high basal levels of this protein by Western-Blot and had mutated p53 (HEL and MV 4.11), whereas its protein expression in the remaining cell lines was low, with KG1 bearing a mutation of the gene and HL60 a p53 deletion. Nevertheless, these last cells with low levels of the protein were a bit more sensitive to the drug, pointing out to a role of p53 in Zalypsis induced cell death. This was in agreement with a clear induction of DNA double strand breaks after treatment with Zalypsis, which was evidenced by an increase in phospho Histone H2AX, phospho CHK1 and phospho CHK2 after treatment of both HEL and HL60 with this compound. This was followed by the overexpression of p53 in the AML cell line bearing low basal levels of this protein (HL60). These results were further confirmed in the gene expression profile studies, in which treatment of AML cells with Zalypsis resulted in an important deregulation of genes involved in DNA damage response, including genes implicated in the ATM repair pathway and other mRNAs related to DNA repair, such as TLK2, ATR, ATMIN, CHEK2, RAD5, RAD52, RAD54L, BRCA1, BRCA2 and GADD45B among others. This response ended up in a clear induction of apoptosis as assessed by annexin V studies, caspase and PARP cleavage with partial rescue of cell death with the caspase inhibitor ZVAD-FMK, DNA laddering and loss of mitochondrial membrane potential analyzed by DiOC6 with the subsequent release of cytochrome C and AIF into the cytosol. Conclusion The potent and selective antileukemic effect observed with Zalypsis in AML cell lines and in patient's samples through a DNA damage response, together with its activity in both p53 mutated- and deleted-cells, provides the rationale for the investigation of Zalypsis in clinical trials for patients with AML. Disclosures: Galmarini: Pharmamar: Employment. Avilés:Pharmamar: Employment. Cuevas:Pharmamar: Employment. Pandiella:Pharmamar: Research Funding. San-Miguel:Pharmamar: Research Funding.

Introduction Plitidepsin is a cyclic depsipeptide isolated from the marine tunicate, Aplidium albicans with promising antitumor activity. This work represents a comprehensive study (in vitro, in vivo and clinical) of its antimyeloma efficacy. Material & Methods In vitro studies were performed in 23 multiple myeloma (MM) cell lines and in cells from 16 MM patients. For the in vivo analysis a human plasmocytoma model in CB17-SCID mouse was used. Mice were randomized to receive Aplidin® 100 μg/Kg ip x 7 days/week (n=9), Aplidin® 140 μg/Kg ip x 5 days/week (n=7) or vehicle alone (n=9). The clinical efficacy of Aplidin® in relapsed/refractory patients was evaluated in a non-randomized two-stage Phase II, multicenter, clinical trial. Dosage of Aplidin® was 5 mg/m2 every 2 weeks. Results Aplidin® showed clear in vitro efficacy (IC50:1–10 nM) in the 23 cell lines tested including those resistant to dexamethasone, melphalan or doxorubicin. It was also active in the presence of microenvironment (IL-6, IGF-1 and BMSCs). Thirteen out of the 16 patient samples were sensitive to Aplidin® with >80% cell death in 8 cases and 60–80% in the remaining ones without significant toxicity in non tumor cells. Combination of Aplidin® with dexamethasone, bortezomib or lenalidomide showed clear potentiation. Aplidin® acts by inducing apoptosis with caspase−3, −7, −8, −9 and PARP cleavage. It also involves the activation of p38 and JNK signalling, Fas/CD95 translocation to lipid rafts and downregulation of Mcl-1 and myc. In mice studies, both schedules of treatment reduced tumor growth and increased survival with statistical differences in the group receiving 140 μg/Kg x 5d/week (p=0.04, Log Rank p=0.02). No significant toxicity was observed. These data provided the rationale for a clinical trial that has included 31 patients with relapsed/refractory MM. Median age was 65 years (47–82) and the median number of prior lines of therapy was 4 (range: 1–9) including autologous stem cell transplant (60%), thalidomide (58%) and bortezomib (48%). Out of the 26 evaluable patients, 2 (8%) achieved PR and 3 (12%) MR. Eight patients (31%) remained in stable disease (SD). Due to the synergism with dexamethasone observed in the in vitro studies, the protocol was amended to allow the addition of this agent in pts progressing after 3 cycles or with SD after 4 cycles. With a median follow-up of 14 months (range: 6.8–16.3), the time to progression in responding pts was 5.8 months (4.9–7.6). The most common G3-4 adverse events were fatigue (7%), serum creatine phosphokinase increase (7%), muscle toxicity (10%) and hepatic toxicity (10%). No significant hematologic toxicity or neuropathy was observed. Conclusion Aplidin® is effective both as a single agent and in combination with dexamethasone in the in vitro and in vivo settings. Its activity in relapsed/refractory MM patients is promising with an acceptable toxicity profile.