Abstract LB319: Nucleolin protein recognition of MYC oncogene promoter G-quadruplex

Abstract

Abstract MYC, one of the most important oncogenes, is a highly validated therapeutic target. However, targeting the MYC protein by small molecules is difficult due to its disordered structure and lack of drug binding pocket. G-quadruplexes (G4s), non-canonical globular DNA secondary structures, form under physiological conditions and are stabilized by cellular K+ or Na+. MYC has a G4-forming region in its proximal promoter region that functions as a transcription silencer. Compounds that bind to and stabilize the MYC promoter G-quadruplex (MycG4) can lower MYC levels in cancer cells. Thus, MycG4 represents an attractive target for small molecules to repress MYC expression. Nucleolin was identified in 2009 as a major MycG4 binding protein that functions as a transcription repressor. Nucleolin’ s best-known substrate is NRE-RNA (nucleolin recognition element) with a stem-loop structure. Remarkably, nucleolin binds the MycG4 with significantly higher affinity than NRE-RNA. However, little is known about how nucleolin recognizes the MycG4. Actually, how G-quadruplex is recognized by a regulatory protein is largely unknown. Here, we determined the crystal structure of the nucleolin protein bound MycG4 complex at 2.6 Å. This is the first high-resolution structure of a G-quadruplex in complex with a regulating protein. Nucleolin contains four tandem RNA-binding domains (RBDs) for nucleic acid interactions. We found that all four RBDs of nucleolin are needed for high-affinity binding with MycG4, in contrast to the binding of NRE-RNA which only requires two RBDs. The free MYC G-quadruplex Myc161 adopts a parallel-stranded three-tetrad structure in K+ solution. Importantly, in the nucleolin-bound form in the crystal structure, the Myc161 remains a parallel-stranded G-quadruplex, exhibiting a very well-defined density with two potassium ions clearly observed between the three G-tetrads. Significantly, a similar 5’-capping DNA-triad structure is observed in the nucleolin bound Myc161 G-quadruplex as well as in the free Myc161 in solution. Using a polymerase extension assay, we showed that nucleolin binding to MycG4 blocks polymerase extension. Furthermore, we performed CUT&Tag sequencing experiments and demonstrated that nucleolin binds to the MYC promoter G4-forming region in cells. In summary, our structure reveals the first G4-conformation-based recognition of a modular protein through multivalent interactions. Our findings indicate that G-quadruplexes are nucleolin's primary cellular substrate and suggest a G4-based epigenetic transcriptional regulation. The nucleolin-MycG4 complex presents a more specific molecular target than MycG4 DNA alone for MycG4-targeted anticancer drug design. Therefore, the nucleolin-MycG4 complex structure will provide important information for understanding MycG4 function and regulation as well as structure-based rational design of MYC-targeted anticancer drugs. Citation Format: Luying Chen, Jonathan Dickerhoff, Ke-wei Zheng, Satchal Erramilli, Guanhui Wu, Saburo Sakai, Danzhou Yang. Nucleolin protein recognition of MYC oncogene promoter G-quadruplex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB319.