PO-473 Quantification of ERCC1-XPF complexes in ovarian cancer xenografts with different sensitivity to cisplatin

Introduction Epithelial ovarian cancer is the most lethal gynaecological cancer due to the development of resistance to a platinum based therapy. As DNA repair capacity is a key determinant for the cellular response to platinum (DDP) agents, DNA repair functional assays are required to study its relevance in DDP resistance. We set up a proximity ligation assay (PLA) to study the activity of nucleotide excision repair (NER) in patient derived ovarian carcinoma xenografts (PDXs) sensitive (S) and resistant (R) to DDP. Material and methods Patient derived xenografts from fresh ovarian carcinomas were recently established in our laboratory. DDP antitumour activity was evaluated in most of the PDXs. Mice were sacrificed when tumours reached 1,5–2 gr. Tumours were fixed in formalin and paraffin embedded (FFPE). PLA was performed on tumour slides, using DuolinkII reagents (Sigma-Aldrich) and following the manufacturer instructions. PLA detects the presence of the protein complexes ERCC1-XPF, that are quantified as foci per nucleus and represent a biomarker of NER activity. Images were acquired by Olympus Virtual Slider (Olympus) and analysed with ImageJ software. Statistical analysis was performed with GraphPad Prism7. Results and discussions Our xenobank comprises PDXs with different response to DDP: MNHOC266 and MNHOC230 are very sensitive to the drug, while MNHOC315 is resistant. We also obtained three sublines resistant to DDP (MNHOC124R, MNHOC124LPR and MNHOC239R) starting from sensitive PDXs (MNHOC124S, MNHOC124LPS and MNHOC239S), after several in vivo drug treatments. Statistically significant higher level of ERCC1-XPF foci could be observed in MNHOC124R and MNHOC124LPR as compared to their sensitive counterparts. No differences were observed between MNHOC239S and R PDXs, even if the number of ERCC1-XPF foci in MNHOC239S were statistically higher than the ones observed in MNHOC124S and in MNHOC124LPS. MNHOC266 and MNHOC230 showed levels of foci comparable to those of MNHOC124S and MNHOC124LPS. mRNA and protein levels of the different isoforms of ERCC1 and of XPF were not different among the PDXs studied. Conclusion PLA for the detection of ERCC1-XPF complexes was set up in FFPE xenograft tumour slides. These preliminary results highlight a possible link between DDP resistance and higher NER activity that need to be confirmed in a wider panel of PDXs. In addition, these data confirm the importance to develop functional assays to directly evaluate the activity of different DNA repair pathways to predict DDP activity

A minimal promoter for TFIIIC-dependent in vitro transcription of snoRNA and tRNA genes by RNA polymerase III.

The Saccharomyces cerevisiae SNR52 gene is unique among the snoRNA coding genes in being transcribed by RNA polymerase III. The primary transcript of SNR52 is a 250-nucleotide precursor RNA from which a long leader sequence is cleaved to generate the mature snR52 RNA. We found that the box A and box B sequence elements in the leader region are both required for the in vivo accumulation of the snoRNA. As expected box B, but not box A, was absolutely required for stable TFIIIC, yet in vitro. Surprisingly, however, the box B was found to be largely dispensable for in vitro transcription of SNR52, whereas the box A-mutated template effectively recruited TFIIIB; yet it was transcriptionally inactive. Even in the complete absence of box B and both upstream TATA-like and T-rich elements, the box A still directed efficient, TFIIIC-dependent transcription. Box B-independent transcription was also observed for two members of the tRNA(Asn)(GTT) gene family, but not for two tRNA(Pro)(AGG) gene copies. Fully recombinant TFIIIC supported box B-independent transcription of both SNR52 and tRNA(Asn) genes, but only in the presence of TFIIIB reconstituted with a crude B'' fraction. Non-TFIIIB component(s) in this fraction were also required for transcription of wild-type SNR52. Transcription of the box B-less tRNA(Asn) genes was strongly influenced by their 5'-flanking regions, and it was stimulated by TBP and Brf1 proteins synergistically. The box A can thus be viewed as a core TFIIIC-interacting element that, assisted by upstream TFIIIB-DNA contacts, is sufficient to promote class III gene transcription

Role of histone modifications and early termination in pervasive transcription and antisense-mediated gene silencing in yeast

Most genomes, including yeast Saccharomyces cerevisiae, are pervasively transcribed producing numerous non-coding RNAs, many of which are unstable and eliminated by nuclear or cytoplasmic surveillance pathways. We previously showed that accumulation of PHO84 antisense RNA (asRNA), in cells lacking the nuclear exosome component Rrp6, is paralleled by repression of sense transcription in a process dependent on the Hda1 histone deacetylase (HDAC) and the H3K4 histone methyl transferase Set1. Here we investigate this process genome-wide and measure the whole transcriptome of various histone modification mutants in a Δrrp6 strain using tiling arrays. We confirm widespread occurrence of potentially antisense-dependent gene regulation and identify three functionally distinct classes of genes that accumulate asRNAs in the absence of Rrp6. These classes differ in whether the genes are silenced by the asRNA and whether the silencing is HDACs and histone methyl transferase-dependent. Among the distinguishing features of asRNAs with regulatory potential, we identify weak early termination by Nrd1/Nab3/Sen1, extension of the asRNA into the open reading frame promoter and dependence of the silencing capacity on Set1 and the HDACs Hda1 and Rpd3 particularly at promoters undergoing extensive chromatin remodelling. Finally, depending on the efficiency of Nrd1/Nab3/Sen1 early termination, asRNA levels are modulated and their capability of silencing is change

Tolerability of Eribulin and correlation between polymorphisms and neuropathy in an unselected population of female patients with metastatic breast cancer: results of the multicenter, single arm, phase IV PAINTER study

Background Metastatic breast cancer (MBC) is an incurable disease and its treatment focuses on prolonging patients' (pts) overall survival (OS) and improving their quality of life. Eribulin is a microtubule inhibitor that increases OS in pre-treated MBC pts. The most common adverse events (AEs) are asthenia, neutropenia and peripheral neuropathy (PN). Methods PAINTER is a single arm, phase IV study, aimed at evaluating the tolerability of eribulin in MBC pts. Secondary objectives were the description of treatment efficacy and safety, the assessment of the incidence and severity of PN and its association with genetic polymorphisms. Genomic DNA was isolated from blood samples and 15 Single Nucleotide Polymorphisms (SNPs) were genotyped by Taqman specific assays. The association between PN and SNPs were evaluated by Fisher exact test. Results Starting from May 2014 until June 2018 180 pts were enrolled in this study by 20 Italian centers. 170 of these pts could be evaluated for efficacy and toxicity and 159 for polymorphisms analysis. The median age of pts was 60 years old and the biological subtypes were luminal type (64.7%), Her2 positive (18.3%) and triple negative (17%). Pts were pretreated with a median of 5 lines for MBC. The median follow up of this study was 15.4 months with a median number of 4.5 cycles administered (minimum-maximum 1-23). The median overall survival was 12 months. 48.8% of pts experienced a dose reduction, mainly for neutropenia (23.9%) and liver toxicity (12%). 65 pts (38.2%) reported at least one severe toxicity. Neutropenia and neurotoxicity were the most frequent severe AEs (15.3% and 14.7%, respectively); other reported toxicities were osteo-muscular, abdominal or tumor site pain (19.4%), liver toxicity (6.6%), pulmonary toxicity (6.5%) and dermatological toxicity (3.6%). Among the 15 evaluated SNPs, an association with PN was found for rs2233335 and rs7214723. Conclusions Eribulin is a well-tolerated treatment option in MBC. Schedule and dosage modifications were common, but toxicity rarely led to treatment discontinuation. SNPs rs2233335 (G/T and T/T) in the NDRG1 gene and rs7214723 (CC and CT) in the CAMKK1 gene were associated with PN. These findings, if validated, could allow a tailored treatment with eribulin in cancer patients. Trial registration: ClinicalTrials.gov ID: NCT02864030

Distal chromatin structure influences local nucleosome positions and gene expression

The positions of nucleosomes across the genome influence several cellular processes, including gene transcription. However, our understanding of the factors dictating where nucleosomes are located and how this affects gene regulation is still limited. Here, we perform an extensive in vivo study to investigate the influence of the neighboring chromatin structure on local nucleosome positioning and gene expression. Using truncated versions of the Saccharomyces cerevisiae URA3 gene, we show that nucleosome positions in the URA3 promoter are at least partly determined by the local DNA sequence, with so-called ‘antinucleosomal elements’ like poly(dA:dT) tracts being key determinants of nucleosome positions. In addition, we show that changes in the nucleosome positions in the URA3 promoter strongly affect the promoter activity. Most interestingly, in addition to demonstrating the effect of the local DNA sequence, our study provides novel in vivo evidence that nucleosome positions are also affected by the position of neighboring nucleosomes. Nucleosome structure may therefore be an important selective force for conservation of gene order on a chromosome, because relocating a gene to another genomic position (where the positions of neighboring nucleosomes are different from the original locus) can have dramatic consequences for the gene's nucleosome structure and thus its expression

Caratterizzazione della qualità e origine delle risorse idriche sotterranee del lodigiano con l'ausilio di strumenti idrochimici ed isotopici

Il lavoro ha avuto come obiettivo la delineazione delle differenze esistenti tra la falda freatica e le falde confinate profonde nel territorio della Provincia di Lodi, attraverso la ricostruzione dell’assetto idrogeologico del sottosuolo e l’ausilio delle tecniche proprie dell’idrochimica e della geochimica isotopica

Characterization of the quality and origin of groundwater of Lodigiano (Northern Italy) with hydrochemical and isotopic instruments

This paper describes the differences between the phreatic aquifer and the deep confi ned aquifers in the territory of Lodi Province (Northern Italy), derived by the reconstruction of the ydrogeological structure of the subsoil and with the aid of hydrochemical and isotopic techniques. Firstly, the presence of a substrate with a marine origin near to the topographic surface infl uences the chemical characteristics of groundwater in some parts of the territory. Secondly, the distribution of the most important chemical parameters, and of some minor chemical parameters with sampling depth, shows that the phreatic aquifer, with a calcium-bicarbonate facies, has a medium-low mineralization, with a decreasing trend in relation to the increase in depth. The isotopic results obtained from the analysis of nitrogen-15 and oxygen-18 in the nitrate molecule permit us to verify the ori- gin of this compound (essentially agricultural), and the occurrence of denitrifi cation phenomena, due to the establishment of a reducing environment at relatively moderate depths, thus testifying to the selfpurifying capacity of the phreatic aquifer with respect to nitrates. Finally, the isotopic investigation on oxygen-18 in the water molecule allows for the identifi cation of different recharge areas and, therefore, the origin of the different aquifers