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.
Promoters often contain asymmetric G- and C-rich strands, in which the cytosines are prone to epigenetic modification via methylation (5-mC) and 5-hydroxymethylation (5-hmC). These sequences can also form four-stranded G-quadruplex (G4) or i-motif (iM) secondary structures. Although the requisite sequences for epigenetic modulation and iM/G4 formation are similar and can overlap, they are unlikely to coexist. Despite 5-hmC being an oxidization product of 5-mC, the two modified bases cluster at distinct loci. This study focuses on the intersection of G4/iM formation and 5-hmC modification using the vascular endothelial growth factor (VEGF) gene promoter's CpG sites and examines whether incorporation of 5-hmC into iM/G4 structures had any physicochemical effect on formation, stability, or recognition by nucleolin or the cationic porphyrin, TMPyP4. No marked changes were found in the formation or stability of iM and G4 structures; however, changes in recognition by nucleolin or TMPyP4 occurred with 5-hmC modification wherein protein and compound binding to 5-hmC modified G4s was notably reduced. G4/iM structures in the VEGF promoter are promising therapeutic targets for antiangiogenic therapy, and this work contributes to a comprehensive understanding of their governing principles related to potential transcriptional control and targeting.
Pancreatic cancer is the fourth most deadly cancer with 5 year survival rate of ∼6%. One of the major attributes of pancreatic cancer is kRAS mutations. kRAS is a proto-oncogene with intrinsic GTPase activity, and is responsible for cell proliferation, division, and apoptosis. kRAS mutations are observed in >95% of pancreatic adenocarcinoma and in 30% all human tumors. When mutated it leads to continuous activity and uncontrolled proliferation which results in increased tumorigenicity and poor prognosis. Downregulating kRAS expression has shown to halt proliferation and leads to cellular death in pancreatic cancer models, but to date no small molecule capable of such transcriptional silencing has been described. A novel series of molecules with either biphenylene or bipyridine spacer connecting the terminal benzofuran ring was synthesized using solution phase chemistry. Tertiary amine side chains were incorporated to the C2-position of the benzofuran ring to improve the DNA binding affinity of these compounds. The synthesized compounds were purified using a “catch and release” method employing the sulfonic acid based resins. The eleven novel compounds were screened in parallel for their ability to stabilize G-quadruplex structures in the kRAS promoter, and to downregulate kRAS promoter activity. Two of the compounds increased the thermal profile of non-canonical DNA formations in the promoter region by >5 °C; however, on the whole these compounds did not seem to function as G-quadruplex-stabilizing agents. Eight compounds significantly decreased luciferase expression under the explicit control of the kRAS promoter by up to 75%, by compound BF 4.3, through an as-yet unknown mechanism of action. The compound series was examined for the inhibition of cellular viability in two mutant kRAS pancreatic cancer cell lines - Panc-1 and MIA PaCa-2; in both cell lines the compounds containing the bipyridine spacer had greater cytotoxic effects with IC50's of ∼30 μM, consistently. qPCR is being used to confirm the decrease in promoter activity in native cellular conditions. These agents are intriguing in both their novel scaffold and their unexpected activity decreasing kRAS expression in a mechanism unrelated to higher order DNA structures. Ultimately, down regulation of kRAS transcription will provide a novel therapeutic approach for pancreatic cancer and improve the prognosis of this highly lethal disease. Citation Format: Harshul Batra, Mohammad K. Islam, David E. Thurston, Khondaker M. Rahman, Tracy A. Brooks. Biphenylene and bipyridine connected benzofuran compounds as novel regulators of kRAS 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 2914.
kRAS is a GTPase protein affecting normal cell proliferation, division, and apoptosis. It is mutated in >30% of all cancers, particularly in 60-90% of pancreatic cancers. Mutated kRAS leads to continuous activity and uncontrolled proliferation. To date there have been no successful clinical agents targeting mutant kRAS activity. Modulating kRAS expression has shown anti-proliferative promise as an approach in pancreatic cancer models, but is not a main avenue of pursuit for clinical development due to lack of a molecular target. Our lab is focused on the regulation of kRAS transcription through a variety of G-rich regions of DNA in the promoter capable of forming non-B-DNA structures termed G-quadruplexes (G4s). In the current study, we sought to determine the effect of the transcription factor MAZ on the regulation of kRAS, with a particular focus on the three putative G4-forming regions (herein termed near, mid, and far in reference to their relative proximity to the transcriptional start site). In a study of the kRAS promoter using a series of luciferase plasmids, MAZ expression led to a concentration-dependent decrease in promoter activity. This action was localized to interactions with the mid-G4-forming region. In the context of a more multifaceted intracellular mileu as found in the pancreatic cancer cell lines MiaPaCa-2, Panc-1, and BxPc3, the effects of MAZ were much more complex. In particular, 48 hr post-transfection with MAZ expression plasmids, monitoring of kRAS transcription revealed a cell line-specific modulation with an increase in kRAS expression in Panc-1 cells only, confirming a previous study, and no changes noted in either the MiaPaCa-2 or the Bx-Pc3 cells. Further studies are ongoing evaluating the direct binding of MAZ to the various G-rich regions in those cell lines, as well as determining the DNA structure (single-stranded, double-stranded, or G4-DNA) to which the MAZ protein is binding. Ultimately, an understanding of the regulation of this G-rich region of DNA, by MAZ or any other transcription factors such as Sp1 or p53, is an important part of the larger puzzle leading to a targeted drug discovery program focused on G4-regulation. Ultimately, G4-stabilization-mediated down regulation of kRAS has high potential for anti-cancer efficacy in pancreatic cancers, where there is a dire need for novel therapeutic development. Citation Format: Harshul Batra, Tracy A. Brooks. The effect of the transcription factor MAZ on kRAS transcription: a role for the G-quadruplex. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2137. doi:10.1158/1538-7445.AM2015-2137
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