Targeting MYC Expression through G-Quadruplexes

Abstract

In this review, the authors describe a novel mechanism for control of MYC expression that involves a four-stranded DNA structure, termed a G-quadruplex, amenable to small molecule targeting. The DNA element involved in this mechanism, the nuclease hypersensitive element III(1) (NHE III(1)), is just upstream of the P1 promoter and is subjected to dynamic stress (negative superhelicity) resulting from transcription. This is sufficient to convert the duplex DNA to a G-quadruplex on the purine-rich strand and an i-motif of the pyrimidine-rich strand, which displaces the activating transcription factors to silence gene expression. Specific proteins have been identified, NM23-H2 and nucleolin, that resolve and fold the G-quadruplex to activate and silence MYC expression, respectively. Inhibition of the activity of NM23-H2 molecules that bind to the G-quadruplex silences gene expression, and redistribution of nucleolin from the nucleolus to the nucleoplasm is expected to inhibit MYC. The authors also describe the mechanism of action of Quarfloxin, a first-in-class G-quadruplex-interactive compound that involves the redistribution of nucleolin from the nucleolus to the nucleoplasm. G-quadruplexes have been best known as test-tube oddities for more than four decades. However, during the past decade, they have emerged as likely players in a number of important biological processes, including transcriptional control. Only time will tell if these odd DNA structures will assume the role of an established receptor class, but it is clear from the scientific literature that there is a dramatic increase in interest in this little-known area in the past few years.

Figure 1.

Building blocks and structures of the G-quadruplex and i-motif located in the NHE III₁ MYC promoter element. Four guanines form a tetrad, and three tetrads form an intramolecularly linked G-quadruplex having a 1:2:1 looping pattern (upper panel). Two sets of cytosine⁺–cytosine base pairs form the basic i-motif unit, and three sets of two cytosine⁺–cytosine base pairs form intramolecularly an i-motif having a 6:2:6 looping pattern (lower panel). Asymmetric positioning of the dimethyl sulfate–protected G-quadruplex and i-motif, with 14- and 5-base overhangs, is indicated by the top and bottom brackets, respectively, in the right central panel. Reprinted from Brooks et al. with permission of the publisher.

Figure 2.

(A) Structures of TMPyP4 and TMPyP2. (B) The rearrangements involved in the Ramos and CA46 Burkitt lymphoma cell lines. Vertical arrows indicate the breakage and rejoining points between chromosomes 14 and 8 for each translocation. (C) RT-PCR for MYC and β-actin in Ramos (lanes 1–3) and CA46 (lanes 4–6) cell lines after no treatment (lanes 1 and 4) and treatment with 100 µM TMPyP2 (P2; lanes 2 and 5) and TMPyP4 (P4; lanes 3 and 6) for 48 hours. Reprinted from Siddiqui-Jain et al. with permission of the publisher.

Figure 3.

(A) A proposed model of MYC transcriptional regulation that involves the resolution of the G-quadruplex by NM23-H2 for duplex DNA formation and subsequent transcriptional activation by Sp1. The binding of hnRNP K and CNBP to single-stranded DNA induced by negative supercoiling also leads to MYC transcription activation. The stabilization of the G-quadruplex by nucleolin results in negative regulation of MYC transcription. (B) The involvement of NM23-H2 and a G-quadruplex-interactive compound in modulating the activation and silencing of the NHE III₁ in the MYC promoter: (a) the G-quadruplex/i-motif form of the NHE III₁, which is the silencer element; (a → c via b) the remodeling of the G-quadruplex/i-motif complex by NM23-H2 in which a stepwise unfolding of the secondary DNA structure is proposed to take place; (b¹) a molecular model of the purine-rich strand unwound from the G-quadruplex form superimposed on an NM23-H2 trimer; (d) the TMPyP4-stabilized G-quadruplex, which prevents NM23-H2 unfolding of the MYC G-quadruplex, Figure 3A reprinted from Kendrick and Hurley; Figure 3B modified from González and Hurley.

Figure 4.

(A) Origin and lead optimization of Quarfloxin (CX-3543). The design of the fluoroquinolone QQ58 was based on the principle that while norfloxacin and A-62176 inhibited gyrase and topoisomerase II by interaction with the enzyme–duplex DNA complex, this scaffold was amenable to G-quadruplex interaction by extending the tetracyclic nucleus to a larger, approximately planar system that would interact more favorably with the tetrad in the G-quadruplex than with a duplex DNA. Cylene Pharmaceuticals then optimized the planar, presumably tetrad-interacting moiety and identified groove-binding arms that would both prevent topoisomerase poisoning and increase selectivity between different G-quadruplexes. (B) The proposed mechanism of action of Quarfloxin involving the MYC G-quadruplex.^, (For the other target proteins of nucleolin, see text.) Quarfloxin is preferentially concentrated in the nucleolus (see inset). Binding of Quarfloxin to the nontemplate G-quadruplexes in the rDNA displaces nucleolin, which is relocated to the nucleoplasm, where it has been shown to bind to the MYC G-quadruplex to inhibit MYC expression.