Targeted downregulation of c-Myc through disruption of the transcription factor/G-quadruplex complex

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Overexpression of the c-Myc oncogene plays a role in carcinogenesis in a variety of cancers, including cervical, breast, and colon, due to dysregulation of cellular growth and apoptosis. Studies have shown that up to 90% of c-Myc promoter activity is tightly controlled by the formation of a G-quadruplex secondary structure in the NHE_III region of this promoter. The formation of the secondary structure silences gene transcription and, subsequently, translation. Using high-throughput screening techniques, such as Fluorescence Resonance Energy Transfer (FRET), a group of compounds was identified that stabilize the c-Myc G-quadruplex structure. One compound (GQC-01) and one of similar structure (GQC-02) decrease fluorescence by over 60%, indicating a stabilization of the G-quadruplex structure. Further ex vivo studies also confirm the FRET findings. By Taq Polymerase Stop assay, GQC-01 and GQC-02 (10 µM) increase stop product by 36% and 47%, respectively. Using a colon cancer model with high c-Myc expression (HCT116), the two compounds of interest were shown to decrease c-Myc gene expression by at least 50%. This decrease translated into a downregulation of protein expression, as determined by Western blot analysis. Exposure of the HCT116 to the compounds for 48 hours resulted in an induction of caspase-3, indicative of apoptosis. Furthermore, electrophoretic mobility shift assays suggest that compound-induced stabilization of the c-Myc G-quadruplex inhibits binding of the regulatory protein NM23-H2 and subsequent unraveling of the G-quadruplex. Together, these data characterize the activity of small molecular agents, with a novel mechanism of transcriptional regulation, that hold great promise for a unique method of anticancer therapy.