Identification Of Effective New Drugs Combinations Exploiting The Ability Of trabectedin To Modulate Transcription

Ewing’s sarcoma (ES) is the second most common malignant bone tumor of childhood, characterized by the chimeric protein EWS-FLI1 (hallmark of this pathology), that alters the transcription of different genes. The five-year overall survival of patients with localized disease is approximately 70%; unfortunately the majority of patients with a metastatic disease have a poor prognosis with a five-year overall survival around the 30%. Among the recently registered drugs for the therapy of sarcomas, trabectedin could be of potential great interest as it seems very active in some “translocated sarcomas”. Trabectedin exerts its antitumor activity with different mechanisms of action. One of the most important is related to its ability to interfere with DNA repair mechanisms (NER and HR), that cause cell cycle perturbations. Furthermore trabectedin is able to displace the oncogenic EWS-FLI1 chimera from its target promoters, modulating the transcription of these genes, in ES cells. Although trabectedin has shown some activity against ES, the overall clinical results indicated only a marginally activity of trabectedin given as single agent in ES. The thesis is aimed at using the available knowledge on trabectedin mechanism of action to identify some effective combinations. Since trabectedin induces cell cycle perturbations I speculated that its activity could be increased by checkpoint inhibitors. The studies performed on the combination of trabectedin and the WEE1 inhibitor, AZD-1775, have shown that the inhibition of WEE1 enhances the trabectedin activity, thus the combination is synergic. Since trabectedin affects the transcription of several genes, I have developed a new approach based on the use of silencing RNA (siRNA) libraries to identify whether the downregulation of some genes was synthetically lethal when ES cells were pretreated with trabectedin. An important part of the thesis was the development, validation and initial application of this approach

A Systems Biology Approach to Characterize the Regulatory Networks Leading to Trabectedin Resistance in an In Vitro Model of Myxoid Liposarcoma

Trabectedin, a new antitumor compound originally derived from a marine tunicate, is clinically effective in soft tissue sarcoma. The drug has shown a high selectivity for myxoid liposarcoma, characterized by the translocation t(12;16)(q13; p11) leading to the expression of FUS-CHOP fusion gene. Trabectedin appears to act interfering with mechanisms of transcription regulation. In particular, the transactivating activity of FUS-CHOP was found to be impaired by trabectedin treatment. Even after prolonged response resistance occurs and thus it is important to elucidate the mechanisms of resistance to trabectedin. To this end we developed and characterized a myxoid liposarcoma cell line resistant to trabectedin (402-91/ET), obtained by exposing the parental 402-91 cell line to stepwise increases in drug concentration. The aim of this study was to compare mRNAs, miRNAs and proteins profiles of 402-91 and 402-91/ET cells through a systems biology approach. We identified 3,083 genes, 47 miRNAs and 336 proteins differentially expressed between 402-91 and 402-91/ET cell lines. Interestingly three miRNAs among those differentially expressed, miR-130a, miR-21 and miR-7, harbored CHOP binding sites in their promoter region. We used computational approaches to integrate the three regulatory layers and to generate a molecular map describing the altered circuits in sensitive and resistant cell lines. By combining transcriptomic and proteomic data, we reconstructed two different networks, i.e. apoptosis and cell cycle regulation, that could play a key role in modulating trabectedin resistance. This approach highlights the central role of genes such as CCDN1, RB1, E2F4, TNF, CDKN1C and ABL1 in both pre- and post-transcriptional regulatory network. The validation of these results in in vivo models might be clinically relevant to stratify myxoid liposarcoma patients with different sensitivity to trabectedin treatment

Toxicological Assessment of ITER-Like Tungsten Nanoparticles Using an In Vitro 3D Human Airway Epithelium Model

International audienceThe International Thermonuclear Experimental Reactor (ITER) is an international project aimed at the production of carbon-free energy through the use of thermonuclear fusion. During ITER operation, in case of a loss-of-vacuum-accident, tungsten nanoparticles (W-NPs) could potentially be released into the environment and induce occupational exposure via inhalation. W-NPs toxicity was evaluated on MucilAir™, a 3D in vitro cell model of the human airway epithelium. MucilAir™ was exposed for 24 h to metallic ITER-like milled W-NPs, tungstate (WO$_4$$^{2−}$) and tungsten carbide cobalt particles alloy (WC-Co). Cytotoxicity and its reversibility were assessed using a kinetic mode up to 28 days after exposure. Epithelial tightness, metabolic activity and interleukin-8 release were also evaluated. Electron microscopy was performed to determine any morphological modification, while mass spectrometry allowed the quantification of W-NPs internalization and of W transfer through the MucilAir™. Our results underlined a decrease in barrier integrity, no effect on metabolic activity or cell viability and a transient increase in IL-8 secretion after exposure to ITER-like milled W-NPs. These effects were associated with W-transfer through the epithelium, but not with intracellular accumulation. We have shown that, under our experimental conditions, ITER-like milled W-NPs have a minor impact on the MucilAir™ in vitro model

Oxidative transformation of Tungsten (W) nanoparticles potentially released in aqueous and biologicalmedia in case of Tokamak (nuclear fusion) Lost of Vacuum Accident (LOVA)

International audienceOxidative transformation of Tungsten (W) nanoparticles potentially released in aqueous and biologicalmedia in case of Tokamak (nuclear fusion) Lost of Vacuum Accident (LOVA) technological breakthroughs need to be achieved before fusion become available and economicallyviable. In addition, and prior to industrial development of the fusion technology, it is worth addressing possible negative environmental and health impacts. For instance, the interactions between the plasma and refractory materials called plasma facing components (PFC) like tungsten, will generate tritiated dust. The aim of the study is to address the fate in water and biological media of W nanoparticles that might be released in case of Lost Of Vacuum Accident (LOVA). The dilution of particles in TRIS, LHC9 and pulmonary media did not strongly affect the average size of the particles while the dilution in Saline medium lead to substantial aggregation. The results proved that oxidative dissolution of W nanoparticles occurred in several aqueous/biological media (TRIS, LHC9 and Lung media) with increasing time. Fromthe different dissolution rates as a function of the tested media, it seems that the oxidative dissolutions are rate limited by diffusion in the oxidized layer surrounding the metallic core of particles. The mechanisms of dissolution involved W4Å and W6Å corroded layers prior to W6Å dissolution. Knowledge provided by these dispersion–dissolution experiments helped to determine the environmental mobility and persistence as well as the bio-durability of these tungsten nanoparticles. As dissolution has potential to influence the toxicity of particles, it is a crucial parameter to consider in the risk assessment of particles

Trabectedin efficacy in Ewing sarcoma Is greatly increased by combination with anti-IGF signaling agents

[EN]Purpose: Goal of this study was to identify mechanisms that limit efficacy of trabectedin (ET-743, Yondelis) in Ewing sarcoma (EWS), so as to develop a clinical applicable combination therapy. Experimental Design: By chromatin immunoprecipitation, we analyzed EWS-FLI1 binding to the promoters of several target genes, such as TGFβR2, CD99, insulin-like growth factor receptor 1 (IGF1R), and IGF1, both in vitro and in xenografts treated with trabectedin or doxorubicin. Combined therapy with trabectedin and anti-IGF1R agents (AVE1642 HAb; OSI-906) was tested in vitro and in xenografts. Results: We confirm that both trabectedin and doxorubicin were able to strongly reduce EWS-FLI1 (both type I and type II) binding to two representative target genes ( TGFβR2 and CD99), both in vitro and in xenografts. However, trabectedin, but not doxorubicin, was also able to increase the occupancy of EWS-FLI1 to IGF1R promoters, leading to IGF1R upregulation. Inhibition of IGF1R either by the specific AVE1642 human antibody or by the dual IGF1R/insulin receptor inhibitor OSI-906 (Linsitinib) greatly potentiate the efficacy of trabectedin in the 13 EWS cell lines here considered as well as in TC-71 and 6647 xenografts. Combined therapy induced synergistic cytotoxic effects. Trabectedin and OSI-906 deliver complementary messages that likely converge on DNA-damage response and repair pathways. Conclusions: Weshowed that trabectedin may not only inhibit but also enhance the binding of EWS-FLI1 to certain target genes, leading to upregulation of IGF1R. We here provide the rationale for combining trabectedin to anti-IGF1R inhibitors.This work was financially supported by the EU project FP7-HEALTH-2011-two-stage, Project ID 278742 EUROSARC; the Italian Association for Cancer Research (Katia Scotlandi—AIRC Project N.14049; C. Garofalo—MFAG N.11584 and M. D'Incalci—AIRC Project N.14658); The Italian Ministry of Research and Education (F.I.R.B. project number: RBAP11884M_005); The Italian Ministry of the Health (67/GR-2010-2319511, CUP G71J12000830001). E. de Álava's laboratory is supported by the AECC (Asociación Española contra el Cáncer), the Ministry of Economy and Competitivity of Spain-FEDER (PI081828, RD06/0020/0059 RD12/0036/0017, PT13/0010/0056, PI110018, ISCIII Sara Borrell postdoc grant CD06/00001), and Fundación Memoria de D. Manuel Solorzano Barruso, Fundación Cris contra el cancer, and Fundación María García Estrada. J.L. Ordóñez is sponsored by the CSIC and the European Social Fund (postdoctoral grant JAE DOC), A.T. Amaral is sponsored by the Fundaçao para a Ciência e Tecnologia, Portugal (fellowship SFRH/BD/69318/2010); and S. Uboldi is recipient of a FIRC Fellowship N.13743

Target genes shared by miR-7, miR-21 and miR-130a.

<p>Nodes represent miRNAs and mRNA gene expression. Red represents gene/miRNA upregulation, green downregulation.</p

Signature validation of miRNA-mRNA and proteins found differentially expressed between 402-91/ET and 402-91 cell line.

<p><b>Panel A</b>. qRT-PCR and Western blot analysis showing differences in the expression levels of let-7e and its downstream targets (CCDN1, E2F5, SEMA4C, HMGA1 and HMGA2). <b>Panel B</b>. qRT-PCR and Western blot for miR-21 and its downstream target, PDCD4. qRT-PCR data are the mean of three independent experiments performed in triplicate and calculated with the 2^−ΔΔCt method as described in the material and method section. The control 402-91 cells values are arbitrarily set as 1. Bars are +/− SD. * is significant with p<0.05, ** p<0.01, ***p<0.001, as assessed with Student T-test. Western blot is representative of at least two independent experiments.</p

Regulatory loops identified through the combination of miRNA, mRNA and protein expression levels.

<p><b>Panel A</b>. Coherent regulatory loops: up/down miRNA regulating the target mRNA through degradation. <b>Panel B</b>. Incoherent regulatory loops: an up/down miRNA regulating the protein level through translational repression. <b>Panel C</b>. Incoherent regulatory loops: an up/down miRNA whose effect is not sufficient to overcome the effect of another external signal and thus it is impossible to hypothesize if miRNA acts as mRNA degradation or translational repressor.</p

Post-transcriptional network.

<p>miRNA and mRNA subnetworks representing negative regulation of apoptosis (Panel A) and cell cycle (Panel B). <u>Small circle</u>: differentially expressed genes found by microarray analysis. <u>Big circle</u>: differentially expressed genes validated by qRT-PCR. <u>Square</u>: differentially expressed genes encoding a protein found differentially expressed using protein array. <u>Exagon</u>: differentially expressed genes validated with qRT-PCR encoding a protein found differentially expressed using protein array. <u>Small diamonds</u>: differentially expressed miRNAs found by array analysis. <u>Big diamonds</u>: differentially expressed miRNAs validated by RT-PCR. Filled colors inside represent gene expression, border colors outside represent protein level.</p