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Background In the search of genetic determinants of Duchenne muscular dystrophy (DMD) severity, LTBP4, a member of the latent TGF-β binding protein family, emerged as an important predictor of functional outcome trajectories in mice and humans. Nonsynonymous single-nucleotide polymorphisms in LTBP4 gene associate with prolonged ambulation in DMD patients, whereas an in-frame insertion polymorphism in the mouse LTBP4 locus modulates disease severity in mice by altering proteolytic stability of the Ltbp4 protein and release of transforming growth factor-β (TGF-β). Givinostat, a pan-histone deacetylase inhibitor currently in phase III clinical trials for DMD treatment, significantly reduces fibrosis in muscle tissue and promotes the increase of the cross-sectional area (CSA) of muscles in mdx mice. In this study, we investigated the activity of Givinostat in mdx and in D2.B10 mice, two mouse models expressing different Ltbp4 variants and developing mild or more severe disease as a function of Ltbp4 polymorphism. Methods Givinostat and steroids were administrated for 15 weeks in both DMD murine models and their efficacy was evaluated by grip strength and run to exhaustion functional tests. Histological examinations of skeletal muscles were also performed to assess the percentage of fibrotic area and CSA increase. Results Givinostat treatment increased maximal normalized strength to levels that were comparable to those of healthy mice in both DMD models. The effect of Givinostat in both grip strength and exhaustion tests was dose-dependent in both strains, and in D2.B10 mice, Givinostat outperformed steroids at its highest dose. The in vivo treatment with Givinostat was effective in improving muscle morphology in both mdx and D2.B10 mice by reducing fibrosis. Conclusion Our study provides evidence that Givinostat has a significant effect in ameliorating both muscle function and histological parameters in mdx and D2.B10 murine models suggesting a potential benefit also for patients with a poor prognosis LTBP4 genotype.

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.

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.

Mass Spectrometry Imaging (MSI) is a widespread technique used to qualitatively describe in two dimensions the distribution of endogenous or exogenous compounds within tissue sections. Absolute quantification of drugs using MSI is a recent challenge that just in the last years has started to be addressed. Starting from a two dimensional MSI protocol, we developed a three-dimensional pipeline to study drug penetration in tumors and to develop a new drug quantification method by MALDI MSI. Paclitaxel distribution and concentration in different tumors were measured in a 3D model of Malignant Pleural Mesothelioma (MPM), which is known to be a very heterogeneous neoplasm, highly resistant to different drugs. The 3D computational reconstruction allows an accurate description of tumor PTX penetration, adding information about the heterogeneity of tumor drug distribution due to the complex microenvironment. The use of an internal standard, homogenously sprayed on tissue slices, ensures quantitative results that are similar to those obtained using HPLC. The 3D model gives important information about the drug concentration in different tumor sub-volumes and shows that the great part of each tumor is not reached by the drug, suggesting the concept of pseudo-resistance as a further explanation for ineffective therapies and tumors relapse.

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.

Polymer nanoparticles (NPs) represent a promising way to deliver poorly water-soluble anticancer drugs without the use of unwanted excipients, whose presence can be the cause of severe side effects. In this work, a Cremophor-free formulation for paclitaxel (PTX) has been developed by employing PEGylated polymer nanoparticles (NPs) as drug delivery carriers based on modified poly(Ε-caprolactone) macromonomers and synthesized through free radical emulsion polymerization. Paclitaxel was loaded in the NPs in a postsynthesis process which allowed to obtain a drug concentration suitable for in vivo use. In vivo experiments on drug biodistribution and therapeutic efficacy show comparable behavior between the NPs and the Cremophor formulation, also showing good tolerability of the new formulation proposed.

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.

One of the major drawbacks that limits the clinical application of nanoparticles is the lack of preliminary investigations related to their biocompatibility, biodegradability and biodistribution. In this work, biodegradable PEGylated polymer nanoparticles (NPs) have been synthesized by using macromonomers based on poly(ε-caprolaconte) oligomers. More in detail, NPs have been produced by adopting a surfactant-free semibatch emulsion polymerization process using PEG chains as a stabilizing agent. The NPs were also labeled with rhodamine B covalently bound to the NPs to quantitatively study their biodistribution in vivo. NPs were investigated in both in vitro and in vivo preclinical systems to study their biodistribution in mice bearing B16/F10 melanoma, as well as their biocompatibility and biodegradability. The NP concentration was evaluated in different tissues at several times after intravenous injection. The disappearance of the NPs from the plasma was biphasic, with distribution and elimination half-lives of 30 min and 15 h, respectively. NPs were retained in tumors and in filter organs for a long time, were still detectable after 7 d and maintained a steady concentration in the tumor for 120 h. 48 h after injection, 70± 15% of the inoculated NPs were excreted in the feces. The favorable tumor uptake, fast excretion and absence of cytotoxicity foster the further development of produced NPs as drug delivery carriers. © 2014 IOP Publishing Ltd.

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.

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.

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.