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Trabectedin (ET743) and lurbinectedin (PM01183) limit the production of inflammatory cytokines that are elevated during cancer cachexia. Mice carrying C26 colon adenocarcinoma display cachexia (i.e., premature death and body wasting with muscle, fat and cardiac tissue depletion), high levels of inflammatory cytokines and subsequent splenomegaly. We tested whether such drugs protected these mice from cachexia. Ten-week-old mice were inoculated with C26 cells and three days later randomized to receive intravenously vehicle or 0.05 mg/kg ET743 or 0.07 mg/kg PM01183, three times a week for three weeks. ET743 or PM01183 extended the lifespan of C26-mice by 30% or 85%, respectively, without affecting tumor growth or food intake. Within 13 days from C26 implant, both drugs did not protect fat, muscle and heart from cachexia. Since PM01183 extended the animal survival more than ET743, we analyzed PM01183 further. In tibialis anterior of C26-mice, but not in atrophying myotubes, PM01183 restrained the NF-&kappa, B/PAX7/myogenin axis, possibly reducing the pro-inflammatory milieu, and failed to limit the C/EBP&beta, /atrogin-1 axis. Inflammation-mediated splenomegaly of C26-mice was inhibited by PM01183 for as long as the treatment lasted, without reducing IL-6, M-CSF or IL-1&beta, in plasma. ET743 and PM01183 extend the survival of C26-bearing mice unchanging tumor growth or cachexia but possibly restrain muscle-related inflammation and C26-induced splenomegaly.

Mass spectrometry imaging (MSI) allows visualization of endogenous and exogenous compound in tissue sections based on its molecular mass. The 3D reconstruction by MSI provides a more informative description of the tumor drug distribution compared to the high-performance liquid chromatography method, highlighting the heterogeneity of intratumor drug concentration. This additional information can be important in understanding chemoresistance to target agents. Here, we present the 3D visualization of the tyrosine kinase inhibitor (TKI), imatinib, in a xenograft model of resistant malignant pleural mesothelioma.

It has recently been reported that a large proportion of human malignant pleural mesothelioma (MPM) cell lines and patient tissue samples present high expression of the c-MYC oncogene. This gene drives several tumorigenic processes and is overexpressed in many cancers. Although c-MYC is a strategic target to restrain cancer processes, no drugs acting as c-MYC inhibitors are available. The novel thienotriazolodiazepine small-molecule bromodomain inhibitor OTX015/MK-8628 has shown potent antiproliferative activity accompanied by c-MYC downregulation in several tumor types. This study was designed to evaluate the growth inhibitory effect of OTX015 on patient-derived MPM473, MPM487 and MPM60 mesothelioma cell lines and its antitumor activity in three patient-derived xenograft models, MPM473, MPM487 and MPM484, comparing it with cisplatin, gemcitabine and pemetrexed, three agents which are currently used to treat MPM in the clinic. OTX015 caused a significant delay in cell growth both in vitro and in vivo. It was the most effective drug in MPM473 xenografts and showed a similar level of activity as the most efficient treatment in the other two MPM models (gemcitabine in MPM487 and cisplatin in MPM484). In vitro studies showed that OTX015 downregulated c-MYC protein levels in both MPM473 and MPM487 cell lines. Our findings represent the first evidence of promising therapeutic activity of OTX015 in mesothelioma.

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

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

The penetration of anticancer drugs in solid tumors is important to ensure the therapeutic effect, so methods are needed to understand drug distribution in different parts of the tumor. Mass spectrometry imaging (MSI) has great potential in this field to visualize drug distribution in organs and tumor tissues with good spatial resolution and superior specificity. We present an accurate and reproducible imaging method to investigate the variation of drug distribution in different parts of solid tumors. The method was applied to study the distribution of paclitaxel in three ovarian cancer models with different histopathological characteristics and in colon cancer (HCT116), breast cancer (MDA-MB-231) and malignant pleural mesothelioma (MPM487). The heterogeneous drug penetration in the tumors is evident from the MALDI imaging results and from the images analysis. The differences between the various models do not always relate to significant changes in drug content in tumor homogenate examined by classical HPLC analysis. The specificity of the method clarifies the heterogeneity of the drug distribution that is analyzed from a quantitative point of view too, highlighting how marked are the variations of paclitaxel amounts in different part of solid tumors.

Background: Trabectedin is an anticancer agent registered for the second-line treatment of soft tissue sarcoma and ovarian cancer. No preclinical data are available on its tumor distribution, so a method for quantification in neoplastic tissues is required. Methods/results: We validated an LC–MS/MS assay determining the recovery, sensitivity, linearity, precision and accuracy in mouse tumor and liver samples. The limit of quantification was 0.10 ng/ml with a curve range of 0.10–3.00 ng/ml (accuracy 96.1–102.1%). Inter-day precision and accuracy of QCs were 6.0–8.2 and 97.0–102.6% respectively. The method was applied in mesothelioma xenografts treated with therapeutic doses. Conclusion: The method was validated for measuring trabectedin in tissues. In a mesothelioma xenograft model, trabectedin distributed preferentially in tumor compared with liver.

To elucidate the mechanisms behind the high sensitivity of myxoid/round cell liposarcoma (MRCL) to trabectedin and the suggested selectivity for specific subtypes, we have developed and characterized three MRCL xenografts, namely ML017, ML015 and ML004 differing for the break point of the fusion gene FUS-CHOP, respectively of type I, II and III. FUS-CHOP binding to the promoters of some target genes such as Pentraxin 3 or Fibronectin 1, assessed by chromatin immunoprecipitation, was strongly reduced in the tumor 24 h after the first or the third weekly dose of trabectedin, indicating that the drug at therapeutic doses causes a detachment of the FUS-CHOP chimera from its target promoters as previously shown in vitro. Moreover, the higher sensitivity of MRCL types I and II appears to be related to a more prolonged block of the transactivating activity of the fusion protein. Doxorubicin did not affect the binding of FUS-CHOP to target promoters. Histologically, the response to trabectedin in ML017 and ML015 was associated with a marked depletion of non-lipogenic tumoral cells and vascular component, as well as lipidic maturation as confirmed by PPARγ2 expression in western Blot. By contrast, in ML004 no major changes either in the cellularity or in the amount of mature were found, and consistently PPARγ2 was null. In conclusion, the data support the view that the selective mechanism of action of trabectedin in MRCL is specific and related to its ability to cause a functional inactivation of the oncogenic chimera with consequent derepression of the adypocytic differentiation.

E-3810 is a novel small molecule that inhibits VEGF receptor-1, -2, and -3 and fibroblast growth factor receptor-1 tyrosine kinases at nmol/L concentrations currently in phase clinical II. In preclinical studies, it had a broad spectrum of antitumor activity when used as monotherapy in a variety of human xenografts. We here investigated the activity of E-3810 combined with different cytotoxic agents in a MDA-MB-231 triple-negative breast cancer xenograft model. The molecule could be safely administered with 5-fluorouracil, cisplatin, and paclitaxel. The E-3810–paclitaxel combination showed a striking activity with complete, lasting tumor regressions; the antitumor activity of the combination was also confirmed in another triple-negative breast xenograft, MX-1. The activity was superior to that of the combinations paclitaxel+brivanib and paclitaxel+sunitinib. Pharmacokinetics studies suggest that the extra antitumor activity of the combination is not due to higher paclitaxel tumor levels, which in fact were lower in mice pretreated with all three kinase inhibitors, and the paclitaxel plasma levels excluded reduced drug availability. Pharmacodynamic studies showed that E-3810, brivanib, and sunitinib given as single agents or in combination with paclitaxel reduced the number of vessels, but did not modify vessel maturation. Reduced tumor collagen IV and increased plasma collagen IV, associated with increased matrix metalloproteinases (MMP), particularly host MMP-9, indicate a proteolytic remodeling of the extracellular matrix caused by E-3810 that in conjunction with the cytotoxic effect of paclitaxel on the tumor cells (caspase-3/7 activity) may contribute to the striking activity of their combination. These data support the therapeutic potential of combining E-3810 with conventional chemotherapy. Mol Cancer Ther; 12(2); 131–40. ©2012 AACR.

Malignant pleural mesothelioma (MPM) incidence is increased over the past two decades and it is predicted to increase further in the next 20 years especially in developing countries where asbestos has not yet been banned. Surgery is not an option for the majority of patients due to the diffuse growth of MPM, making chemotherapy the treatment of choice, but prognosis remains very poor with a median survival time of 10-17 months. Trabectedin (ET-743, Yondelis) is a marine alkaloid used for the 2nd line therapy of ovarian cancer (combined with pegylated doxorubicin) and soft tissue sarcomas. It binds the DNA minor groove modulating transcription of cancer cells and has an anti-inflammatory effect modifying tumor microenvironment and reducing tumor associated macrophages. The aim was to test antitumor activity of trabectedin in newly established patient derived xenografts of MPM. MPM473, MPM484 and MPM487 xenografts were obtained in athymic nude mice by s.c. injection of 107 cells isolated from the pleural effusion of MPM patients and maintained by serial passages in mice. H&E staining and immunohistochemistry were performed. Mice were randomized when tumor reached about 200 mg to receive trabectedin, cisplatin, pemetrexed, gemcitabine, and imatinib. Antitumor activity was expressed as T/C% were T and C are the mean tumor weight of treated and control mice, respectively. The three xenografts reproduced the morphology and the histochemical profile of the human tumors. As reported in the table trabectedin was able to induce a 50% reduction in tumor growth in MPM473 and MPM 487 xenografts and only a marginal effect in MPM484 xenograft. TreatmentDose (mg/kg)ScheduleBest T/C% (Day)MPM473MPM484MPM487Cisplatin5i.v. q7dx361.9 (69)66.1 (69)67.3 (39)Gemcitabine100i.p. q4dx583.2 (41)67.7 (48)49.7 (56)Imatinib200p.o. qdx1568.9 (76)89.4 (55)87.1 (39)Pemetrexed250i.p. q4dx477.4 (52)76.8 (49)84.3 (39)Trabectedin0.15i.v. q7dx351.0 (73)63.6 (69)51.3 (56) In conclusion we report three new MPM xenografts able to mimic the pathological features of human MPM. Although trabectedin showed only a moderate antitumor activity in our experimental models, its efficacy is comparable or better than that of the other drugs currently used for this disease. Studies are in progress in syngenic models that are more appropriate to test also the ability of trabectedin to modulate the immune system. Citation Format: Simonetta Andrea Licandro, Roberta Frapolli, Ezia Bello, Roberta Libener, Sara Orecchia, Federica Grosso, Maurizio D’Incalci. Trabectedin activity in patient-derived mesothelioma xenografts. [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 3764.

Trabectedin (ET-743, Yondelis) is a marine alkaloid isolated from the tunicate Ecteinascidia turbinata, approved in Europe and US for the 2nd line therapy of soft tissue sarcomas. It is particularly effective against myxoid liposarcoma, a specific histological subtype within the family of adults soft tissue sarcomas. More than 90% of usual myxoid/round cell liposarcomas (MRCLS) are characterized by the chromosomal translocation t(12;16) (q13; p11), which produces the FUS-CHOP oncogene. Different chimera subtypes seem to respond differently to trabectedin in clinical setting. Trabectedin was able to remove FUS-CHOP type I, II and III from its own target genes but it causes adipocytic maturation and tumor regression only in type I and II, not in type III MRCLS (Di Giandomenico et al, Oncogene, 2014 Oct 30; 33(44): 5201-10). Pioglitazone is a PPARgamma agonist with pro-differentiation effects in different cancer types included liposarcomas (Tontonoz P. et al, Proc Natl Acad Sci U S A. 1997 Jan 7;94(1): 237-41). The aim of the study is to evaluate the efficacy of the combination of trabectedin with pioglitazone in type III myxoid liposarcoma xenografts. ML006 and ML004 type III MRCLS xenografts mice were treated with trabectedin 0.15 mg/kg iv every seven days for three times or pioglitazone 150 mg/kg po daily for 28 days or with their combination. For antitumor activity evaluation, tumor growth was measured with a caliper and the tumor weights (1 mm3 = 1 mg) were calculated with the formula: length × (width)2/2. Fifteen days after the last dose of trabectedin and 4 h after the last dose of pioglitazone tumors were collected from a separate group of mice to perform histological and molecular analyses. Trabectedin and pioglitazone as single agents have a comparable antitumor activity on these models with T/C around 40-50% with no tumor regression observed. In the combination groups an impressive and long lasting tumor regression was observed in ML 006 xenograft, with an observed optimal T/C of 17%. In ML004 MRCLS model the combination of trabectedin and pioglitazone induced a minimal regression followed by a long lasting tumor stabilization, resulting in a T/C of 23%. The histological analyses showed adipocytic maturation in tumors treated with pioglitazone alone or in combination but not in samples from mice treated with trabectedin alone. In conclusion, the combination of trabectedin with pioglitazone was able to induce tumor regression and adipocytic maturation in type III myxoid liposarcoma xenografts that were only marginally sensitive to trabectedin alone. Chromatin Immunoprecipitation, gene expression profile and Western Blot analysis are on-going to unravel the molecular mechanism behind the potentiation of the antitumor efficacy observed with this combination. Citation Format: Ezia Bello, Roberta Frapolli, Simonetta Andrea Licandro, Silvia Brich, Laura Carrassa, Roberta Sanfilippo, Alessandro Gronchi, Paolo Casali, Silvana Pilotti, Maurizio D’Incalci. PPARgamma agonist promotes adipocytic differentiation and potentiates the activity of trabectedin in myxoid liposarcoma. [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 1183.

library were cultured for 13 days, after which shRNAs were recovered by PCR. Deep sequencing was applied to determine the relative abundance of each shRNA in BRAF (V600E) cells as compared to WT2 CC cells. Results and Conclusions: Based on the results of the pooled shRNA screen, we were able to identify six candidate synthetic lethal genes in BRAF mutant CC cell lines. In particular, further validation showed RANBP2 gene knock-down to be synthetic lethal with BRAFV600E and BRAFlikeness in CC. Experiments addressing the identification and the biological characterization of RANBP2 will be presented.

RNA Pol II in global NER (XPC) deficient cells, but failed to do it in TC NER (CSB, XPD and XPG) deficient cells. Importantly, these effects were confirmed to be specific for the Rpb1 subunit of RNA Pol II, since other subunits were not affected (Rpb2 and Rpb4) as well as other factors of the transcriptional machinery, such as TBP (TFIID), p62 (TFIIH), XPD or the RPA194 subunit of the RNA Pol I. Finally, it was also demonstrated that, contrary to what occurs after DNA damage with UV light, the transcription of p53 target genes important for DNA repair, including p21 or mdm2, was irreversibly inhibited after PM01183 treatment. Together, these results show the mechanism by which PM01183 inhibits trans-activated transcription process on tumor cells.

Mesothelioma, sometimes referred to as asbestos cancer, is known to be among the most aggressive and difficult to treat tumours. The disease attacks the mesothelium, a protective two-layered membrane that covers the internal organs of the body including the lungs, heart and abdominal organs. Mesothelioma can affect any of these layers, but is usually seen in the pleural (lung) or peritoneal (abdomen) mesothelium. The most commonly diagnosed form of this cancer is pleural mesothelioma, caused primarily by the inhalation of asbestos fibers. The prevalence of this cancer is estimated at less than 1% of all cancers, however its incidence is increasing, with an expected peak in the next 10-20 years. Mesotheliomas are tumours that have a poor prognosis due to limited treatment options. The most aggressive form is the sarcomatoid histotype, followed by the biphasic and the epithelioid histotypes.In order to setup in vivo models of human mesothelioma, translational related to human pathology in terms of histology, antigen expression and pharmacological response to therapy, we have established three mesothelioma in vivo models using primary cells derived from patients having different mesothelioma histotypes.We stably transfected MM-432 (sarcomatoid), MM-473 (epithelioid) and MM-487 (biphasic) mesothelioma cell lines with a luciferase expression vector. The selected high luc-espressing clones were inoculated intrapleurally in immunodeficient nude mice, and they successfully invaded and proliferated within the murine host. In particular, the epithelioid histotype presented tumour growth 100% of the time, after a short latency period. The biphasic histotype failed to present tumour growth in 30% of the cases, however was far more aggressive than the epithelial histotype upon establishment of tumour growth. Moreover, the ex-vivo Immunocytochemistry (i.e for CEA, EMA, Mesothelin, Podoplanin, calretinin) and biochemical characterization (i.e.EGFR, VEGF, Top1, TKs pathways) of the three histotypes, revealed histotype-related differences. Finally, the cytotoxic responses to a panel of antitumor drugs (i.e . Doxorubicin , Topotecan , Ciclofosfamide, Dacarbazina, Gemcitabine, Temozolomide, Bortezomib, 5-azacitidina, Paclitaxel, Etoposide, 5-FU, Cisplatin, and AZD-2281) revealed that the three original cell lines, compared to the corresponding luc-expressing clones, had similar sensitivities to drugs depending upon the tumor histotypes. Further, in vivo characterizations focused on chemotherapeutic responses of these models, are currently in progress. Citation Format: Claudio Pisano, Alison G. Cole, Marcella Barbarino, Erminia Bianchino, Mario Guglielmi, Carmela Melito, Grazia Mercadante, Alfonsina Porciello, Assunta Riccio, Ilaria La Porta, Sara Orecchia, Roberta Libener, Laura Mazzucco, Simonetta Andrea Licandro, Pasquale De Luca. Mice models of three human mesothelioma histotypes derived from primary patient tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5556. doi:10.1158/1538-7445.AM2014-5556

Introduction: Trabectedin (T) is a marine-derived anticancer drug with a peculiar mechanism of action, ascribed to a covalent modification of DNA by guanine-specific alkylation at the N2 position in the minor groove that induces DNA damage, affects transcription regulation and triggers cell differentiation and death. Trabectedin has been approved in the EU for the second line treatment of soft tissue sarcomas and ovarian carcinomas. In order to investigate the mechanisms of resistance to trabectedin, that generally occurs after prolonged treatment with the drug, two different trabectedin-resistant cell lines such as myxoid liposarcoma 402-91 and ovarian carcinoma A2780 were developed and characterized. Material and Methods: Resistant cell lines (402-91/T and A2780/T) were obtained by stepwise increase in drug concentration using a short exposure (1 hr) applied at 10-20 day intervals. Drug sensitivity was measured by colony assay, DNA content and cell cycle were evaluated by flow cytometric analysis and XPG expression by Western Blotting. Results: 402-91/T and A2780/T cell lines were 16 and 6 fold resistant to trabectedin as compared to parental cells respectively. Resistant cell lines maintained the same DNA content compared with the parental cells, while showed different cell cycle perturbations induced by trabectedin treatment; in fact the characteristic G2/M block was found only in sensitive cell lines. Both 402-91/T and A2780/T cell lines did not express XPG, a protein involved in NER pathway, and were found hypersensitive to UV light. This finding is in keeping with previous observations that NER deficiency is associated to a decreased sensitivity to trabectedin. Being NER activity crucial for the repair of platinum DNA adducts, we tested cisplatin cytotoxicity in 402-91/T and A2780/T cell lines and they were 2.5 and 11 fold more sensitive than the parental cell lines. The collateral sensitivity to cisplatin of trabectedin resistant cell lines is likely related to the important function of NER in the repair of the bulky adducts produced by the drug. Conclusion: The finding that resistance to trabectedin is associated to the loss of NER function, with consequent increased sensitivity to cisplatin, provides the rational to test the sequential combination of trabectedin and platinum complexes in the clinic. This strategy is currently under evaluation in ovarian cancer patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C93. Citation Format: Maurizio D'Incalci, Benedetta Colmegna, Sarah Uboldi, Roberta Frapolli, Simonetta Andrea Licandro, Eugenio Erba, Carlos M. Galmarini, Nadia Badri. Collateral sensitivity to cisplatin of trabectedin-resistant cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C93.