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

Abstract

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.

G-quadruplex–R-loop interactions and the mechanism of anticancer G-quadruplex binders

“…By studying the effects of a naphthalene diimide derivative, CM03 (Figure 2 ), using an RNA-seq approach on two pancreatic cancer cell lines, Marchetti et al ( 151 ) showed after 6 h treatments a large down-regulation of many genes, which are rich in PQS and involved in essential pathways of pancreatic cancer cell survival and tumor progression. In another study ( 152 ), transcriptional expression profiles affected by the G4 binder AQ1 (Figure 2 ), an anthraquinone derivative developed to target the KIT promoter, were determined using an RNA-seq approach in canine and human cell lines. The findings confirmed the KIT gene expression down-regulation but also the down-regulation of MYC-related pathways and up-regulation of p53, apoptosis and hypoxia-response pathways in both species.…”

G-quadruplexes Sequester Free Heme in Living Cells

“…Two G4 ligands analyzed, AQ1 and CM03, are structurally distinct from one another and from PhenDC3 (Figure 5B). AQ1 is an anthraquinone derivative which can displace an end-stacking compound from quadruplexes but does not preferentially act at telomeric structures (Zorzan et al, 2016), and which has been shown to affect proliferation of the mast cell leukemia line HMC1.2 (Zorzan et al, 2018). CM03 is a trisubstituted naphthalene diimide derivative shown to function as a potent inhibitor of proliferation of pancreatic tumor cell lines and xenografts (Marchetti et al, 2018).…”

“…The results we present validate displacement of G4-bound heme by PhenDC3 in vitro and demonstrate, by RNA-seq analysis, that PhenDC3 treatment significantly upregulates pathways involved in heme metabolism and iron homeostasis in human HT1080 fibrosarcoma cells, most strikingly causing a 30-fold induction of HMOX1, the chief enzyme in the heme degradation pathway. A search of publicly available transcriptome datasets showed that a similar restricted subset of genes is induced by treatment with hemin (Ghosh et al, 2011) and with a structurally unrelated G4 ligand, the anthraquinone derivative AQ1 (Zorzan et al, 2018). Thus, G-quadruplexes may maintain genomic integrity by sequestering heme, thereby contributing to the regulation of free heme levels in living cells both locally and genome wide.…”

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

“…Accumulating evidence suggests that these higher order arrangements might work as regulatory elements to control gene transcription [ 9 , 10 ]. In the case of KIT , the G4s formation at the promoter has been associated with a reduction in the corresponding coded protein expression [ 11 , 12 ]. The high-resolution structures of kit1, kit2 and kit* G4 have been solved [ 6 , 7 , 13 , 14 , 15 , 16 ], thus paving the way to a rational design of new ligands able to stabilize them and, ultimately, to silence the gene in the cell.…”

“…After the identification and characterization of all the mutants, we performed site-directed mutagenesis experiments in which the above mentioned libraries were introduced into a plasmid in which luciferase expression was under the control of the wild type (WT) KIT promoter sequence. The domain that resulted more relevant for gene transcription was finally considered as a target of two G4 binders (AQ1 and AN6, Figure S1 ) that were previously confirmed to bind the KIT promoter and to reduce c-kit expression in cells [ 11 , 24 ].…”