Targeting Canine KIT Promoter by a Candidate DNA G-quadruplex Ligand

Introduction. G-quadruplexes (G4) are DNA secondary structures formed by stacked G-tetrads frequently located in promoter regions of proto-oncogenes. The promoter of canine proto-oncogene KIT (a tyrosine kinase receptor), known to be a primary driver of canine mast cell tumor (MCT), contains two G-rich sequences folding into G4. Hence, we aimed to: (1) characterize the transcriptional effects of a candidate G4 ligand known to specifically stabilize human and canine KIT; (2) verify the existence of cross-talks between KIT and other genes along canine transcriptome (e.g., BCL2). Materials and Methods. In a canine MCT cell line (C2), the constitutive expression of KIT and other proto-oncogenes possessing G4 structures (BCL2, VEGFα, MYC, KRAS, TERT) was assessed by quantitative Real time RT-PCR (qPCR). The cytotoxicity (IC50) of an anthraquinone derivative (AQ1) was determined by Alamar Blue test, and its time- and dose-dependent effects upon KIT and aforementioned proto-oncogenes mRNA levels were evaluated by qPCR. To confirm if AQ1 effectively binds to KIT proximal promoter, confirmatory dual-luciferase bioassays were executed using Madin-Darby Canine Kidney (MDCK) cells. Based on the obtained results, possible transcriptional cross-talks between KIT and the whole canine transcriptome were checked on C2 cells exposed to AQ1 by using an Illumina RNAseq technology. Results and Conclusions. AQ1 significantly decreased cell proliferation, KIT mRNA and c-kit protein amount; however, this downregulation did not result from a specific interaction with KIT proximal promoter. The RNAseq analysis showed a dose-dependent increasing number of down-regulated genes, including anti-apoptotic genes (i.e., BCL2) and other proto-oncogenes (e.g., FYN and EGFR). Overall, AQ1 showed a minor efficacy in canine C2 cells compared to human cell lines (HMC1.2). Actually, a bisantrene derivative (AN6) has already been tested in C2 cells and seems to be a further promising G4 ligand to efficiently target canine KIT. Acknowledgements Project supported by University of Padova (CPDA147272, DOR1798331)