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 \u201cin house\u201d 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\u2013dependent cell lines that present either c-KIT mutations or promoter engineered (i.e., \u3b1155, 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

Histone acylations and chromatin dynamics: Concepts, challenges, and links to metabolism.

In eukaryotic cells, DNA is tightly packed with the help of histone proteins into chromatin. Chromatin architecture can be modified by various post-translational modifications of histone proteins. For almost 60 years now, studies on histone lysine acetylation have unraveled the contribution of this acylation to an open chromatin state with increased DNA accessibility, permissive for gene expression. Additional complexity emerged from the discovery of other types of histone lysine acylations. The acyl group donors are products of cellular metabolism, and distinct histone acylations can link the metabolic state of a cell with chromatin architecture and contribute to cellular adaptation through changes in gene expression. Currently, various technical challenges limit our full understanding of the actual impact of most histone acylations on chromatin dynamics and of their biological relevance. In this review, we summarize the state of the art and provide an overview of approaches to overcome these challenges. We further discuss the concept of subnuclear metabolic niches that could regulate local CoA availability and thus couple cellular metabolisms with the epigenome

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

Targeting canine c-KIT promoter by a candidate DNA G-quadruplex ligand

Introduction G-quadruplexes (G-quad) are DNA secondary structures formed by stacked G-tetrads frequently located in telomeres and promoter regions of proto-oncogenes. Recently, two G-rich sequences, canine kit1 (d_kit1) and kit2 (d_kit2), folding into G-quad, have been identified in canine c-KIT promoter. Accordingly, the aim of the present study was to test a possible small molecule inhibitor known to decrease significantly target gene expression by stabilizing KIT1 and KIT2 G-quad structures. Materials and Methods The constitutive gene expression of c-KIT and other proto-oncogenes (BCL2, VEGF\u3b1, VEGFR2, KRAS, TERT) mRNA was measured in canine mast cell tumor (MCT) cell line C2 by quantitative RT-PCR (qPCR). Therefore, the G-quad ligand 50% inhibitory concentration (IC50) was determined by using the Alamar Blue cytotoxicity test. Finally, its time- and dose-dependent transcriptional effects upon c-KIT and other target genes were evaluated by using qPCR. Results In C2 cell line, target genes were shown to be constitutively expressed and measurable up to 96 hours of culture. The IC50 value of G-quad ligand was shown to be 1,37 \ub5M and, at lower cytotoxic concentration, it significantly down-regulated c-KIT mRNA levels (after 24h hours of incubation). A similar effect, although of low intensity, was noticed for BCL2. Conclusions The G-quad candidate ligand seems to be a promising candidate for targeting KIT-dependent tumors such as MCT. However, such an assumption needs to be confirmed with further molecular studies. Comparative studies about the efficacy of this and other G-quad ligands in blocking human kit G-quadruplex are currently underway. Supporting grants: University of Padua (CPDA114388

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, VEGF alpha, 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

Characterization of the promoter region of the proto-oncogene c-kit in canine mast cell tumour

G-quadruplex structures at telomeric and promotorial sequences represent promising chemotherapeutic targets. Small molecules can bind and stabilize already formed G-quadruplexes or can induce their formation from G-rich sequences. In order to select efficient and selective G-quadruplex ligands, the conformational equilibria of the nucleic acid need to be characterized. In this work we are interested in the promotorial sequence of c-kit, a proto-oncogene which over expression or gain of function mutations characterizes human cancers, such as gastrointestinal stromal tumors (GIST), melanoma and mastocytosis. In humans, this region contains two G-rich sequences, c-kit1 and c-kit2. The G-quadruplex structures assumed by these two sequences have been extensively studied by means of X-ray crystallography and NMR. The stabilization of these secondary structures resulted in a suppression of gene transcription. In the domestic dog, c-kit over expression or mutations have been found in cutaneous mast cell tumor (MCT), representing the most common (7%-21%) canine skin tumor.. Therefore, we expect that dog could be a robust model to have a better insight into c-kit abnormalities in cancer and into the role of potential c-kit G-quadruplex ligand in anticancer chemotherapy. To support this, the canine upstream promotorial sequence was cloned and sequenced in both healthy and MCT-suffering dogs. Our results identified two putative G-quadruplex sequences in the canine promotorial region. Thus, we compared them with the human ones. In particular, large attention was devoted to clarify the conformational equilibria occurring in physiologically relevant conditions by means of spectroscopic and electrophoretic techniques. This work represents the required preliminary step for a better understanding of MCT biology, progression and treatment as well as to assess dogs as a translational model for the many c-kit related human tumours

Impairment of c-kit expression in human cancer cell lines by a novel pharmacophoric unit selected for the recognition of the proto-oncogene KIT promotorial region.

G-quadruplex structures at telomeric region and in oncogene promotorial sequences represent promising chemotherapeutic targets. Among these latter ones, an interesting site of intervention is represented by the promotorial sequence of KIT, a proto-oncogene whose overexpression or mutations characterize several human cancers. This region contains two G-rich sequences, kit1 and kit2, whose G-quadruplex structures have been extensively studied by using X-ray crystallography and NMR. The stabilization of these secondary structures by small molecules has been shown to result in a suppression of gene transcription. The peculiarity of these G-quadruplex structures rest in the presence of unprecedented structural domains which foresees the possibility to design small molecules able to recognize them, with a higher selectivity in comparison to other nucleic acid tetra-helixes. In order to identify novel ligands for these sequences, we started a screening program by using an available \u201cin house\u201d library of compounds. Each member was previously confirmed to provide a basal level of nucleic acid recognition. Ligand selection was performed by applying two validated different screening assays (FRET and FID); they were properly designed to identify the best binders and to preliminary exclude compounds with low selectivity among different G-quadruplexes. For selected compounds, the DNA binding properties were fully characterized in terms of binding affinity, binding mode and ability to convert unfolded sequences into a G-quadruplex structure. On parallel, the ligands cellular effects were explored in two human cancer cell lines (MCF7 and HCG27). At first, we evaluated their cytotoxic potential; then, we examined the extent of potential c-kit down-regulation following the exposure to candidates at both mRNA and protein level. By merging these results we were able to highlight the relevance of one pharmacophore as potential selective KIT targeting agent

Succinylation of H3K122 destabilizes nucleosomes and enhances transcription.

Histone post-translational modifications (PTMs) are key players in chromatin regulation. The identification of novel histone acylations raises important questions regarding their role in transcription. In this study, we characterize the role of an acylation on the lateral surface of the histone octamer, H3K122 succinylation (H3K122succ), in chromatin function and transcription. Using chromatin succinylated at H3K122 in in vitro transcription assays, we show that the presence of H3K122succ is sufficient to stimulate transcription. In line with this, we found in our ChIP assays H3K122succ enriched on promoters of active genes and H3K122succ enrichment scaling with gene expression levels. Furthermore, we show that the co-activators p300/CBP can succinylate H3K122 and identify sirtuin 5 (SIRT5) as a new desuccinylase. By applying single molecule FRET assays, we demonstrate a direct effect of H3K122succ on nucleosome stability, indicating an important role for histone succinylation in modulating chromatin dynamics. Together, these data provide the first insights into the mechanisms underlying transcriptional regulation by H3K122succ

Histone propionylation is a mark of active chromatin

Histones are highly covalently modified, but the functions of many of these modifications remain unknown. In particular, it is unclear how histone marks are coupled to cellular metabolism and how this coupling affects chromatin architecture. We identified histone H3 Lys14 (H3K14) as a site of propionylation and butyrylation in vivo and carried out the first systematic characterization of histone propionylation. We found that H3K14pr and H3K14bu are deposited by histone acetyltransferases, are preferentially enriched at promoters of active genes and are recognized by acylation-state-specific reader proteins. In agreement with these findings, propionyl-CoA was able to stimulate transcription in an in vitro transcription system. Notably, genome-wide H3 acylation profiles were redefined following changes to the metabolic state, and deletion of the metabolic enzyme propionyl-CoA carboxylase altered global histone propionylation levels. We propose that histone propionylation, acetylation and butyrylation may act in combination to promote high transcriptional output and to couple cellular metabolism with chromatin structure and function