Identification and characterization of a new G-quadruplex forming region within the kRAS promoter as a transcriptional regulator

Abstract

kRAS is one of the most prevalent oncogenic aberrations. It is either upregulated or mutationally activated in a multitude of cancers, including pancreatic, lung, and colon cancers. While a significant effort has been made to develop drugs that target kRAS, their clinical activity has been disappointing due to a variety of mechanistic hurdles. The presented works describe a novel mechanism and molecular target to downregulate kRAS expression--a previously undescribed G-quadruplex (G4) secondary structure within the proximal promoter acting as a transcriptional silencer. There are three distinct guanine-rich regions within the core kRAS promoter, including a previously examined region (G4near). Of these regions, the most distal region does not form an inducible and stable structure, whereas the two more proximal regions (termed near and mid) do form strong G4s. G4near is predominantly a tri-stacked structure with a discontinuous guanine run incorporated; G4mid consists of seven distinct runs of continuous guanines and forms numerous competing isoforms, including a stable three-tetrad stacked mixed parallel and antiparallel loop structures with longer loops of up to 10 nucleotides. Comprehensive analysis of the regulation of transcription by higher order structures has revealed that the guanine-rich region in the middle of the core promoter, termed G4mid, is a stronger repressor of promoter activity than G4near. Using the extensive guanine-rich region of the kRAS core promoter, and particularly the G4mid structure, as the primary target, future drug discovery programs will have potential to develop a potent, specifically targeted small molecule to be used in the treatment of pancreatic, ovarian, lung, and colon cancers.

Insight into the Complexity of the i-Motif and G-Quadruplex DNA Structures Formed in the KRAS Promoter and Subsequent Drug-Induced Gene Repression

“…As previously reported, 28 the Near-G forms an all-parallel G-quadruplex, while the Mid-G and Far-G form mixed parallel/antiparallel G-quadruplexes, with the Far-G being much weaker than the Mid-G, similar to our analyses of the i-motifs. CD spectral analysis of the three G-quadruplex-forming sequences in 100 mM KCl shows that at 1 and 2 equiv Nitidine increases the molar ellipticity at 260 nm for the Near-G (Figure 7A) and at 295 nm for the Mid-G and Far-G (Figure 7, B and C).…”

“…The presence of three tandem G-quadruplex/i-motif-forming sequences in the KRAS promoter begs the question of why they are all conserved with at least two configurations having been shown to be important in KRAS transcription control. 28 We propose that the population dynamics of the three DNA secondary structure-forming regions could provide a “fail-safe” mechanism to regulate transcription and prevent over-expression of KRAS, as the protein product of this gene plays an important role in numerous cellular processes, including cell proliferation, differentiation, and survival. 71 We propose a model for the functional roles of the DNA secondary structure-mediated control of KRAS transcription (Figure 9).…”

“…On the basis of their proximity to the transcription start site, they are termed the Near-region (−129 to −159), Mid-region (−174 to −228), and Far-region (−238 to −275) (Figure 1A). 28 (Regions on the G-rich strand will be referred to as Near-G, Mid-G, and Far-G while regions on the C-rich strand will be referred to as Near-C, Mid-C, and Far-C).…”

“…28–33 The C-rich region of the murine KRAS promoter was one of the first oncogene promoter i-motifs to be characterized. 34 …”

From basic researches to new achievements in therapeutic strategies of KRAS-driven cancers

“…G-to-T knockout mutations in the G4-forming regions of the KRAS promoter were found to disrupt or abrogate G4 formation. In addition, stabilization of the KRAS promoter by the cationic porphyrin TMPyP4 leads to a significant decrease in KRAS expression 180 . The interaction of G4 of the KRAS promoter with natural polyphenols, such as ellagic acid and curcumin, has also been confirmed by UV-vis spectroscopy.…”