Abstract 3089: A novel G-quadruplex formed in the PDGFR-β promoter that is selectively targeted by a small molecule to repress transcription

Abnormal expression of PDGFR-β protein contributes to several malignancies. PDGFR-β has become increasingly attractive therapeutic target in the treatments of certain cancers. A G-quadruplex-forming nuclease hypersensitive element (NHE) in the human PDGFR-β promoter has been found to form multiple G-quadruplexes from the overlapping sequences. This G-quadruplex-forming NHE has been shown to regulate approximately 60% of basal promoter activity. Targeting transcriptional control of PDGFR-β provides an attractive target for developing inhibitors for the PDGFR-β signaling pathway, in addition to molecular targeting of the PDGFR-β protein or its cognate ligand. We have previously determined the most stable G-quadruplex formed in the PDGFR-β NHE. Interestingly, the 3′-end G-quadruplex formed in the PDGFR-β promoter NHE appears to be selectively targeted by an ellipticine analog GSA1129, which has been shown to repress PDGFR-β activity in cancer cell lines, and GSA1129 appears to shift the dynamic equilibrium in the full-length sequence to favor this structure. Therefore, characterization of the 3′-end G-quadruplex structure is important for understanding its function and for rational design of small molecules targeting this element. The 3′-end PDGFR-β G-quadruplex appears to adopt an unusual parallel G-quadruplex structure containing an imperfect GGGA tetrad at the 3′-end, as shown by DMS footprinting and CD spectroscopy. We further investigated the stability and structure of the 3′-end G-quadruplex and its interactions with GSA1129 by mutational analysis combined with NMR spectroscopy. The 3′-end G-quadruplex can be stabilized by an A-to-G mutation at position 18. However, the truncated wild-type and mutated 18-mer (Pu18) sequences appear to form predominantly dimer structure as shown by NMR. Using a 19-mer sequence Pu19 with the A-to-G mutation (Pu19A18G), a stable monomeric G-quadruplex can be formed in potassium solution, adopting a parallel-stranded structure as shown by NMR. In the wild-type 3’-end NHE sequences, the formation of a monomeric G-quadruplex in potassium can only be observed in a 20-mer Pu20 sequence with additional 3’ G20; the assignment of Pu20 G-quadruplex using site-specific 15N-G-labeled DNA sequences indicated a novel parallel-stranded G-quadruplex structure using G20 instead of A18 in the 3’-end-tetrad. The Pu20 G-quadruplex appears to have high thermo-stability as shown by CD studies; however, its specific formation can only be achieved at low potassium salt. GSA1129 can bind Pu20 and increase the 3′-end G-quadruplex stability by nearly 20 degrees. This study highlighted the dynamic nature of the 3′-end PDGFR-β G-quadruplex and the importance of identifying the proper sequence for the biologically relevant G-quadruplex structure formation. Significantly, the dynamic nature of the 3′-end G-quadruplex may make it an attractive target for drug regulation. Citation Format: Buket Onel, Prashansa Agrawal, Megan Carver, Robert Brown, Laurence Hurley, Danzhou Yang. A novel G-quadruplex formed in the PDGFR-β promoter that is selectively targeted by a small molecule to repress transcription. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3089.