Hypermethylation-Mediated Silencing of CIDEA, MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies.

Gene expression is controlled by epigenetic deregulation, a hallmark of cancer. The DNA methylome of canine diffuse large B-cell lymphoma (cDLBCL), the most frequent malignancy of B-lymphocytes in dog, has recently been investigated, suggesting that aberrant hypermethylation of CpG loci is associated with gene silencing. Here, we used a multi-omics approach (DNA methylome, transcriptome and copy number variations) combined with functional in vitro assays, to identify putative tumour suppressor genes subjected to DNA methylation in cDLBCL. Using four cDLBCL primary cell cultures and CLBL-1 cells, we found that CiDEA, MAL and PCDH17, which were significantly suppressed in DLBCL samples, were hypermethylated and also responsive (at the DNA, mRNA and protein level) to pharmacological unmasking with hypomethylating drugs and histone deacetylase inhibitors. The regulatory mechanism underneath the methylation-dependent inhibition of those target genes expression was then investigated through luciferase and in vitro methylation assays. In the most responsive CpG-rich regions, an in silico analysis allowed the prediction of putative transcription factor binding sites influenced by DNA methylation. Interestingly, regulatory elements for AP2, MZF1, NF-kB, PAX5 and SP1 were commonly identified in all three genes. This study provides a foundation for characterisation and experimental validation of novel epigenetically-dysregulated pathways in cDLBCL.

G-Quadruplex Modulation of SP1 Functional Binding Sites at the KIT Proximal Promoter.

The regulation of conformational arrangements of gene promoters is a physiological mechanism that has been associated with the fine control of gene expression. Indeed, it can drive the time and the location for the selective recruitment of proteins of the transcriptional machinery. Here, we address this issue at the KIT proximal promoter where three G-quadruplex forming sites are present (kit1, kit2 and kit*). On this model, we focused on the interplay between G-quadruplex (G4) formation and SP1 recruitment. By site directed mutagenesis, we prepared a library of plasmids containing mutated sequences of the WT KIT promoter that systematically exploited different G4 formation attitudes and SP1 binding properties. Our transfection data showed that the three different G4 sites of the KIT promoter impact on SP1 binding and protein expression at different levels. Notably, kit2 and kit* structural features represent an on-off system for KIT expression through the recruitment of transcription factors. The use of two G4 binders further helps to address kit2-kit* as a reliable target for pharmacological intervention.

Significance of the goby Zosterisessor ophiocephalus as a sentinel species for Venice Lagoon contamination: Combining biomarker responses and bioaccumulation.

The Venice Lagoon is an interesting example of an ecosystem suffering for a considerable anthropogenic impact, resulting in high concentrations of persistent organic pollutants (POPs) in lagoon sediments and seafood. In this context, biomonitoring is a crucially important task. The present study aimed at evaluating the validity of a multiple biomarker approach in a benthic fish species. A total of 567 Zosterisessor ophiocephalus (Gobiidae) fish were collected in spring and autumn from three areas of Venice Lagoon (Porto Marghera, Val di Brenta, and Cà Roman) showing high, intermediate and low amounts of POPs, respectively. Aryl hydrocarbon receptor (AHR) and cytochrome P450 1A (CYP1A) mRNA levels, CYP1A protein amount and ethoxyresorufin O-deethylase activity (EROD) were measured in pooled liver and gills (mRNA levels only). Such biological data were then compared with polychlorinated biphenyls (PCBs) residues, measured in grass goby muscle by gas chromatography. Aryl hydrocarbon receptor and CYP1A mRNAs, protein and EROD were upregulated in accordance with PCB amounts measured in Z. ophiocephalus muscles. In fact, the highest AHR and CYP1A induction was observed in fish sampled in close proximity of the industrial area of Porto Marghera. Overall, the present study confirm the grass goby as a reliable sentinel species for Venice Lagoon, and AHR/CYP1A/EROD as a sensitive set of biomarkers of exposure for AHR ligands.

Validation of epigenetic mechanisms regulating gene expression in canine B-cell lymphoma: An in vitro and in vivo approach.

Despite canine B-cell Lymphoma (BCL) representing the most common haematological tumour, epigenetic events driving development and progression are scarcely known. Recently, canine Diffuse Large BCL (DLBCL) DNA methylome by genome-wide CpG microarray has identified genes and pathways associated to pathogenesis. To validate data previously obtained by array analysis, the CLBL-1 cell line was used and the HOXD10, FGFR2, ITIH5 and RASAL3 genes were selected. CLBL-1 cells were treated with two hypomethylating drugs (HDs; IC50, 50% inhibitory concentration), i.e. azacytidine and decitabine (DEC), either alone or in combination with three histone deacetylase inhibitors (HDACis; IC20), i.e. valproic acid, trichostatin and vorinostat. Following the incubation with both HDs, an overall decrease of promoter methylation was highlighted, thus confirming target genes hypermethylation. The highest mRNA restoration was observed following the exposure to HDs combined with HDACis, and mostly with valproic acid. Contrasting results were only obtained for RASAL3. An in vivo confirmation was finally attempted treating Nod-Scid mice engrafted with CLBL-1 cells with DEC. Although DEC did not arrest tumour growth, target genes promoter methylation was significantly reduced in DEC-treated mice vs controls. Overall, this work demonstrates that CLBL-1 cell line represents a reliable in vitro model to validate the methylation-dependent silencing of key genes for BCL; moreover, it may be useful for xenograft models in mice, despite its aggressive behaviour. In future, functional studies will be performed to deepen the role of selected genes on BCL pathogenesis and progression, and their methylation-dependent mechanism of regulation.

Whole-Transcriptome Profiling of Canine and Human in Vitro Models Exposed to a G-Quadruplex Binding Small Molecule.

G-quadruplexes (G4) are secondary nucleic acid structures that have been associated with genomic instability and cancer progression. When present in the promoter of some oncogenes, G4 structures can affect gene regulation and, hence, represent a possible therapeutic target. In this study, RNA-Seq was used to explore the effect of a G4-binding anthraquinone derivative, named AQ1, on the whole-transcriptome profiles of two common cell models for the study of KIT pathways; the human mast cell leukemia (HMC1.2) and the canine mast cell tumor (C2). The highest non-cytotoxic dose of AQ1 (2 µM) resulted in 5441 and 1201 differentially expressed genes in the HMC1.2 and C2 cells, respectively. In both cell lines, major pathways such as cell cycle progression, KIT- and MYC-related pathways were negatively enriched in the AQ1-treated group, while other pathways such as p53, apoptosis and hypoxia-related were positively enriched. These findings suggest that AQ1 treatment induces a similar functional response in the human and canine cell models, and provide news insights into using dogs as a reliable translational model for studying G4-binding compounds.

Targeting Canine KIT Promoter by Candidate DNA G-Quadruplex Ligands.

G-quadruplexes (G4) are nucleic acid secondary structures frequently assumed by G-rich sequences located mostly at telomeres and proto-oncogenes promoters. Recently, we identified, in canine KIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog) promoter, two G-rich sequences able to fold into G4: d_kit1 and d_kit2_A16. In this study, an anthraquinone (AQ1) and an anthracene derivative (AN6), known to stabilize the G4 structures of the corresponding human h_kit1 and h_kit2, were tested on the canine G4 and in two canine mast cell tumor (MCT) cell lines (C2 and NI-1) to verify their capability to down-regulate KIT expression. The cytotoxicity of AQ1 and AN6 was determined using the Alamar Blue test; also the constitutive expression of KIT and other proto-oncogenes containing G4 structures in their promoter (BCL2, VEGFα, VEGFR2, KRAS, and TERT) was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). Then the time- and dose-dependent effects of both ligands on target gene expression were assessed by qRT-PCR. All target genes were constitutively expressed up to 96 hours of culture. Both ligands decreased KIT mRNA levels and c-kit protein amount, and AN6 was comparatively fairly more effective. DNA interaction studies and a dual-luciferase gene reporter assay performed on a noncancerous canine cell line (Madin-Darby Canine Kidney cells) proved that this down-regulation was the result of the interaction of AN6 with KIT proximal promoter. Interestingly, our results only partially overlap with those previously obtained in human cell lines, where AQ1 was found as the most effective compound. These preliminary data might suggest AN6 as a promising candidate for the selective targeting of canine KIT-dependent tumors.

Screening of candidate G-quadruplex ligands for the human c-KIT promotorial region and their effects in multiple in-vitro models.

Stabilization of G-quadruplex (G4) structures in promoters is a novel promising strategy to regulate gene expression at transcriptional and translational levels. c-KIT proto-oncogene encodes for a tyrosine kinase receptor. It is involved in several physiological processes, but it is also dysregulated in many diseases, including cancer. Two G-rich sequences able to fold into G4, have been identified in c-KIT proximal promoter, thus representing suitable targets for anticancer intervention. Herein, we screened an "in house" library of compounds for the recognition of these G4 elements and we identified three promising ligands. Their G4-binding properties were analyzed and related to their antiproliferative, transcriptional and post-transcriptional effects in MCF7 and HGC27 cell lines. Besides c-KIT, the transcriptional analysis covered a panel of oncogenes known to possess G4 in their promoters.From these studies, an anthraquinone derivative (AQ1) was found to efficiently downregulate c-KIT mRNA and protein in both cell lines. The targeted activity of AQ1 was confirmed using c-KIT-dependent cell lines that present either c-KIT mutations or promoter engineered (i.e., α155, HMC1.2 and ROSA cells).Present results indicate AQ1 as a promising compound for the target therapy of c-KIT-dependent tumors, worth of further and in depth molecular investigations.

Mutational Hotspot of TET2, IDH1, IDH2, SRSF2, SF3B1, KRAS, and NRAS from Human Systemic Mastocytosis Are Not Conserved in Canine Mast Cell Tumors.

INTRODUCTION: Both canine cutaneous mast cell tumor (MCT) and human systemic mastocytosis (SM) are characterized by abnormal proliferation and accumulation of mast cells in tissues and, frequently, by the presence of activating mutations in the receptor tyrosine kinase V-Kit Hardy-Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog (c-KIT), albeit at different incidence (>80% in SM and 10-30% in MCT). In the last few years, it has been discovered that additional mutations in other genes belonging to the methylation system, the splicing machinery and cell signaling, contribute, with c-KIT, to SM pathogenesis and/or phenotype. In the present study, the mutational profile of the Tet methylcytosine dioxygenase 2 (TET2), the isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2), the serine/arginine-rich splicing factor 2 (SRSF2), the splicing factor 3b subunit 1 (SF3B1), the Kirsten rat sarcoma viral oncogene homolog (KRAS) and the neuroblastoma RAS viral oncogene homolog (NRAS), commonly mutated in human myeloid malignancies and mastocytosis, was investigated in canine MCTs. METHODS: Using the Sanger sequencing method, a cohort of 75 DNA samples extracted from MCT biopsies already investigated for c-KIT mutations were screened for the "human-like" hot spot mutations of listed genes. RESULTS: No mutations were ever identified except for TET2 even if with low frequency (2.7%). In contrast to what is observed in human TET2 no frame-shift mutations were found in MCT samples. CONCLUSION: Results obtained in this preliminary study are suggestive of a substantial difference between human SM and canine MCT if we consider some target genes known to be involved in the pathogenesis of human SM.

Sequencing and G-quadruplex folding of the canine proto-oncogene KIT promoter region: might dog be used as a model for human disease?

Downregulation of gene expression by induction of non-canonical DNA structures at promotorial level is a novel attractive anticancer strategy. In human, two guanine-rich sequences (h_kit1 and h_kit2) were identified in the promotorial region of oncogene KIT. Their stabilization into G-quadruplex structures can find applications in the treatment of leukemias, mastocytosis, gastrointestinal stromal tumor, and lung carcinomas which are often associated to c-kit mis-regulation. Also the most common skin cancer in domestic dog, mast cell tumor, is linked to a mutation and/or to an over-expression of c-kit, thus supporting dog as an excellent animal model. In order to assess if the G-quadruplex mediated mechanism of regulation of c-kit expression is conserved among the two species, herein we cloned and sequenced the canine KIT promoter region and we compared it with the human one in terms of sequence and conformational equilibria in physiologically relevant conditions. Our results evidenced a general conserved promotorial sequence between the two species. As experimentally confirmed, this grants that the conformational features of the canine kit1 sequence are substantially shared with the human one. Conversely, two isoforms of the kit2 sequences were identified in the analyzed dog population. In comparison with the human counterpart, both of them showed an altered distribution among several folded conformations.

DNA and RNA isolation from canine oncologic formalin-fixed, paraffin-embedded tissues for downstream "-omic" analyses: possible or not?

Formalin-fixed, paraffin-embedded (FFPE) tissues represent a unique source of archived biological material, but obtaining suitable DNA and RNA for retrospective "-omic" investigations is still challenging. In the current study, canine tumor FFPE blocks were used to 1) compare common commercial DNA and RNA extraction kits; 2) compare target gene expression measured in FFPE blocks and biopsies stored in a commercial storage reagent; 3) assess the impact of fixation time; and 4) perform biomolecular investigations on archival samples chosen according to formalin fixation times. Nucleic acids yield and quality were determined by spectrophotometer and capillary electrophoresis, respectively. Quantitative real-time polymerase chain reaction assays for the following genes: BCL-2-associated X protein, B-cell lymphoma extra large, antigen identified by monoclonal antibody Ki-67, proto-oncogene c-KIT (c-kit). Two internal control genes (Golgin A1 and canine transmembrane BAX inhibitor motif containing 4), together with direct sequencing of c-kit exons 8, 9, 11, and 17, were used as end points. Differences in DNA/RNA yield and purity were noticed among the commercial kits. Nucleic acids (particularly RNA) extracted from paraffin blocks were degraded, even at lower fixation times. Compared to samples held in the commercial storage reagent, archived tissues showed a poorer amplification. Therefore, a gold standard protocol for DNA/RNA isolation from canine tumor FFPE blocks for molecular investigations is still troublesome. More standardized storage conditions, including time between sample acquisition and fixation, fixation time, and sample thickness, are needed to guarantee the preservation of nucleic acids and, then, their possible use in retrospective transcriptomic analysis.