Deregulation c-MYC is considered to be the hallmark of cancer as it regulates a large array of key genes essential for cell function such as proliferation, metabolism, differentiation, adhesion and apoptosis. Consequently, c-MYC expression is tightly regulated at the transcriptional and post-transcriptional levels. One of the key regulators of c-MYC transcription is the Nuclease Hypersensitive Element III1 (NHEIII1) located up-stream of P1 promoter. NHEIII1 is a gene silencer requiring for its function a stable spatial conformation that is provided by the high G/C rich ratio that forms G-quadruplex and i-motif structures. The oligonucleotide sequence Pu27 (27 nucleotides) forms G-quadruplex and correspond to the genomic NHEIII1 sequence. We have shown that Pu27 reduces c-MYC transcription in leukemia cell lines and consequently inhibits cell growth and promotes apoptosis. In this study, we evaluated the effect of Pu27 modification using polyethylene glycol (PEG), tocopherol (Toco) and the lipid palmitate (Palmi) in order to increase G-quadruplex stability and lessen blood clearance. We investigated the binding property to NHEIII1 target sequence using electrophoretic mobility shift assay (EMSA). The target used for this study was a 134bp covering NHEIII1 as double stranded (ds) and C-rich or G-rich single stranded (ss) DNA. We verify the presence of G-quadruplex formation in the modified Pu27 using circular dichroism (CD) spectrometry in two different salinity conditions 100mM KCl: that favors G-quadruplex formation and 100mM NaCl that does not. In addition, the effect of the modified-Pu on cell proliferation was investigated in U937 and Raji leukemia cell lines using MTT assay. We show that Pu27 binds specifically to the target sequence on the C-rich strand. The CD spectra confirm the presence of G-quadruplex in the modified Pu27. Furthermore, the CD measurement in two different salinity conditions revealed that Toco and Palmi- modifications stabilize the formation of G-quadruplex even in less favorable condition (100mM NaCl). The dose response study in MTT assay reveals that Toco and Palmi modified-Pu inhibit cell growth for Raji and U937 leukemia cell lines as well, if not better than unmodified Pu27. This data demonstrates the specific binding of the G-quadruplex lipid-modified Pu27 at the silencer element in a sequence specific manner (by strand invasion) and down regulation of c-MYC expression as is the case for unmodified Pu27. Subsequently down regulation of c-MYC by Pu27 was followed by change in c-MYC target genes expression and inhibition of cell growth. Our finding suggests that modification of the c-MYC targeted oligonucleotide by addition of lipids stabilizes the 3D structure (G-quadruplex) and improve its function at inhibiting cell growth most likely by down-regulating c-MYC.

Citation Format: Francine Francine, Shelia D. Thomas, Gilles Tapolsky, Donald M. Miller. Lipid modification of c-MYC promoter targeted oligonucleotide stabilizes G-quadruplex formation and enhances its growth inhibitory activity in leukemia cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5418. doi:10.1158/1538-7445.AM2015-5418