Nuclease Hypersensitive Element Research Articles

Design, synthesis, and biological evaluation of novel benzimidazolyl isoxazole derivatives as potential c-Myc G4 stabilizers to suppress c-Myc transcription and myeloma growth

The c-Myc oncogene is one of the most frequently deregulated driver genes in human cancer, and its rearrangement is closely associated with the development of multiple myeloma (MM). However, c-Myc is a disordered protein that lacks adequate binding pockets on its surface and has a half-life of only 20 to 30 minutes, so it is challenging to design small-molecule inhibitors. The guanine-rich nuclease hypersensitive element III1 (NHEIII1) upstream of the P1 promoter of the c-Myc gene can form intramolecular parallel G-quadruplexes (G4) structures. It has been established that c-Myc G4 controls 85-90% of the transcriptional activation of the c-Myc, which represents one of the most sought-after drug targets in cancer. Therefore, targeting c-Myc G4 to inhibit c-Myc protein would be a potential strategy for the treatment of MM. Herein, a series of benzimidazolyl isoxazole derivatives (EP1-EP19) were designed and synthesized. Among them, compound EP12 exhibited better RPMI-8226 cell inhibition activity (IC50 = 6.16 µM). Moreover, compound EP12 induced apoptosis and inhibited the expression of c-Myc mRNA and c-Myc protein at 5 µM. Circular dichroism (CD) and molecular dynamics (MD) simulation studies indicated that compound EP12 could firmly stabilize c-Myc G4. Accordingly, compound EP12 displayed potent anti-MM activities in vitro and might be a potential c-Myc G4 small molecular stabilizer to warrant further investigation.

Structure of two G-quadruplexes in equilibrium in the KRAS promoter.

KRAS is one of the most mutated oncogenes and still considered an undruggable target. An alternative strategy would consist in targeting its gene rather than the protein, specifically the formation of G-quadruplexes (G4) in its promoter. G4 are secondary structures implicated in biological processes, which can be formed among G-rich DNA (or RNA) sequences. Here we have studied the major conformations of the commonly known KRAS 32R, or simply 32R, a 32 residue sequence within the KRAS Nuclease Hypersensitive Element (NHE) region. We have determined the structure of the two major stable conformers that 32R can adopt and which display slow equilibrium (>ms) with each other. By using different biophysical methods, we found that the nucleotides G9, G25, G28 and G32 are particularly implicated in the exchange between these two conformations. We also showed that a triad at the 3′ end further stabilizes one of the G4 conformations, while the second conformer remains more flexible and less stable.

High resolution crystal structure of a KRAS promoter G-quadruplex reveals a dimer with extensive poly-A π-stacking interactions for small-molecule recognition.

Aberrant KRAS signaling is a driver of many cancers and yet remains an elusive target for drug therapy. The nuclease hypersensitive element of the KRAS promoter has been reported to form secondary DNA structures called G-quadruplexes (G4s) which may play important roles in regulating KRAS expression, and has spurred interest in structural elucidation studies of the KRAS G-quadruplexes. Here, we report the first high-resolution crystal structure (1.6 Å) of a KRAS G-quadruplex as a 5′-head-to-head dimer with extensive poly-A π-stacking interactions observed across the dimer. Molecular dynamics simulations confirmed that the poly-A π-stacking interactions are also maintained in the G4 monomers. Docking and molecular dynamics simulations with two G4 ligands that display high stabilization of the KRAS G4 indicated the poly-A loop was a binding site for these ligands in addition to the 5′-G-tetrad. Given sequence and structural variability in the loop regions provide the opportunity for small-molecule targeting of specific G4s, we envisage this high-resolution crystal structure for the KRAS G-quadruplex will aid in the rational design of ligands to selectively target KRAS.

Truncated G-Quadruplex Isomers Cross-Talk with the Transcription Factors To Maintain Homeostatic Equilibria in c-MYC Transcription.

The nuclease hypersensitive element III1 (NHE III1) upstream c-MYC promoter harbors a transcription-silencing G-quadruplex (Pu27) element. Dynamic turnover of various transcription factors (TFs) across Pu27 to control c-MYC transcription homeostasis is enigmatic. Here, we reveal that native Pu27 evolves truncated G-quadruplex isomers (Pu19, Pu22, Pu24, and Pu25) in cells that are optimal intracellular targets of specific TFs in a sequence- and structure-dependent manner. Nuclear magnetic resonance and isothermal titration calorimetry envisaged that NM23-H2 (nucleoside diphosphate kinase) and nucleolin induce conformational fluctuations in Pu27 to sample specific conformationally restricted conformer(s). Structural investigations revealed that the flanking guanines at 5'-Pu27 control solvent exposure at G-quartets upon NM23-H2 and nucleolin binding driving Pu27 unfolding and folding, respectively. Transient chromatin immunoprecipitations confirmed that NM23-H2 drives the conformation switch to Pu24 that outcompetes nucleolin recruitment. Similarly, nucleolin arrests Pu27 in the Pu22 conformer minimizing NM23-H2 binding at Pu27. hnRNPK (heterogeneous nuclear ribonucleoprotein K) positively regulates NM23-H2 and nucleolin association at Pu27 despite their antagonism. On the basis of these results, we simulated the transcription kinetics in a feed-forward loop in which the transcription output responds to hnRNPK-induced early activation via NM23-H2 association, which favors Pu24 formation at NHE III1 reducing nucleolin occupancy and driving quadruplex unfolding to initiate transcription. NM23-H2 further promotes hnRNPK deposition across NHE III1 altering Pu27 plasticity that finally enriches the nucleolin abundance to drive Pu22 formation and weaken NM23-H2 binding to extinguish transcription. This mechanism involves three positive feedback loops (NM23-H2-hnRNPK, NM23-H2-CNBP, and hnRNPK-nucleolin) and one negative feedback loop (NM23-H2-nucleolin) controlling optimal turnover and residence time of TFs at Pu27 to homeostatically regulate c-MYC transcription.

Abstract 685: Structural study of the 3'-end G-quadruplex formed in the human PDGFR-β promoter: Insight into a transcriptional inhibitor element

Abstract Aberrant expression of PDGFR-β promotes multiple hallmarks of cancer, and its transcriptional regulation provides an attractive means to inhibit the PDGFR-β signaling pathway. The human PDGFR-β promoter nuclease hypersensitive element (NHE) can form multiple G-quadruplexes. While the most stable G-quadruplex formed in the human PDGFR-β promoter NHE region is the 5'-mid G-quadruplex, the 3'-end sequence that contains a 3'-GGA run forms a less stable G-quadruplex. Interestingly, the G-quadruplex formed in the 3'-end region of the NHE is recently found to be a transcriptional repressor of PDGFR-β, and a small molecule ellipticine, GSA1129, is shown to selectively stabilize the 3'-end G-quadruplex and repress PDGFR-β activity in cancer cell lines and in a preclinical animal model. Therefore, understanding the molecular structure of the 3'-end G-quadruplex is important for small molecule targeting in PDGFR-β gene regulation. Using Nuclear Magnetic Resonance (NMR) spectroscopy, we found that the 3'-end G-quadruplex formed in the extended PDGFR-β NHE 3'-end sequence consists of two novel intramolecular end-insertion G-quadruplexes, one with a 3'-non-adjacent flanking guanine inserted into the 3'-external tetrad (3'-insertion-G4), and another with a 5'-non-adjacent flanking guanine inserted into the 5'-external tetrad (5'-insertion-G4), respectively. The two guanines in the GGA-run move up or down within the G-quadruplex to accommodate the inserted guanine. The two end-insertion G-quadruplexes co-exist in equilibrium under physiological salt conditions. In the 3'-insertion-G4, the 3' non-adjacent guanine G20 is involved in the formation of the 3'-external G-tetrad, with the two guanines from the GGA-run involved in the formation of the 5'-external and middle G-tetrads, thereby inducing the formation of a bulge loop structure from the 3'-flanking strand. In the 5'-insertion-G4, the 5'-non-adjacent flanking guanine G0 is involved in the formation of the 5'-external G-tetrad, with the two guanines from the GGA-run involved in the formation of the middle and 3'-external G-tetrads, thereby inducing the formation of a lateral loop from the 5'-flanking residues on top of the 5'-external tetrad. The loop interactions and capping structures of the two end-insertion G-quadruplexes appear to be different. For the 3'-insertion-G4, only the 5'-flanking bases stabilize the structure. For the 5'-insertion-G4, the flanking residues at both the 5'- and 3'-ends stabilize the structure, and a favorable capping structure appears to be adopted by the 5'-lateral loop. Significantly, the formation of two equilibrating end-insertion G-quadruplexes appears to depend on the GGA-run and the guanine-containing flanking sequences. The unique end-insertion structures and capping conformations may provide a specific platform for small molecule targeting. Citation Format: Buket Onel, Clement Lin, Danzhou Yang. Structural study of the 3'-end G-quadruplex formed in the human PDGFR-β promoter: Insight into a transcriptional inhibitor element [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 685.

The 3′-end region of the human PDGFR-β core promoter nuclease hypersensitive element forms a mixture of two unique end-insertion G-quadruplexes

BackgroundWhile the most stable G-quadruplex formed in the human PDGFR-β promoter nuclease hypersensitive element (NHE) is the 5′-mid G-quadruplex, the 3′-end sequence that contains a 3′-GGA run forms a less stable G-quadruplex. Recently, the 3′-end G-quadruplex was found to be a transcriptional repressor and can be selectively targeted by a small molecule for PDGFR-β downregulation. MethodWe use 1D and 2D high-field NMR, in combination with Dimethylsulfate Footprinting, Circular Dichroism Spectroscopy, and Electrophoretic Mobility Shift Assay. ResultsWe determine that the PDGFR-β extended 3′-end NHE sequence forms two novel end-insertion intramolecular G-quadruplexes that co-exist in equilibrium under physiological salt conditions. One G-quadruplex has a 3′-non-adjacent flanking guanine inserted into the 3′-external tetrad (3′-insertion-G4), and another has a 5′-non-adjacent flanking guanine inserted into the 5′-external tetrad (5′-insertion-G4). The two guanines in the GGA-run move up or down within the G-quadruplex to accommodate the inserted guanine. Each end-insertion G-quadruplex has a low thermal stability as compared to the 5′-mid G-quadruplex, but the selective stabilization of GSA1129 shifts the equilibrium toward the 3′-end G-quadruplex in the PDGFR-β NHE. ConclusionAn equilibrium mixture of two unique end-insertion intramolecular G-quadruplexes forms in the PDGFR-β NHE 3′-end sequence that contains a GGA-run and non-adjacent guanines in both the 3′- and 5′- flanking segments; the novel end-insertion structures of the 3′-end G-quadruplex are selectively stabilized by GSA1129. General significanceWe show for the first time that an equilibrium mixture of two unusual end-insertion G-quadruplexes forms in a native promoter sequence and appears to be the molecular recognition for PDGFR-β downregulation.

1H, 13C, and 15N chemical shift assignments of a G-quadruplex forming sequence within the KRAS proto-oncogene promoter region.

Single stranded guanine rich DNA (or RNA) sequences adopt noncanonical secondary structures called G-quadruplexes (G4). Functionally, quadruplexes control gene transcription and regulate activities such as replication, gene recombination or alternative splicing. Hence they are potential targets for cancer, neuronal, and viral related diseases. KRAS is one of the most mutated oncogenes in the genome of cancer cells and contains a nuclease hypersensitive element (NHE) sequence capable of forming G-quadruplexes via its six runs of guanines. In our work, we are interested in the NMR structure of the major G4 scaffold formed in the KRAS NHE region with a mutated sequence of 22 residues. Here, we report 1H, 13C and 15N chemical shift assignments the G4 formed within KRAS22RT sequence.

Fluorescent light-up acridine orange derivatives bind and stabilize KRAS-22RT G-quadruplex

KRAS is often found mutated in lethal cancers and should be an important target for anticancer drug development. However, no effective inhibitor has been reported so far, prompting the scientific community to describe the RAS proteins as nearly “undruggable”. Recent approaches developed to modulate KRAS protein expression comprises the targeting of G-quadruplex (G4) structures formed within the nuclease hypersensitive element of KRAS promoter region, by designing small and specific ligands to stabilize the tertiary fold and reduce gene expression. In this work, we report in vitro and in silico studies of novel acridine orange (AO) derivatives (C3-C8), developed as G4 stabilizing agents. The results show that the ligands bind with high affinity and stabilize KRAS22-RT G4 with modest specificity over duplex DNA. The most promising ligand C8 stabilizes the structure by ≈ 40 °C. Molecular docking using NMR-derived distance restraints reveal atomic details about the ligand structural features in the interaction with KRAS22-RT G4. In vitro studies with HeLa cells show that the ligands are cytotoxic with IC50 values between 0.9 μM and 5.7 μM. Moreover, the ligands tend to localize in the nucleus as shown by confocal fluorescence microscopy. Overall, these results show that the reported AO ligands display favourable properties as G4 ligands and this study provides structural detail for the development of lead KRAS G4 ligands.

In Silico Screening and Binding Characterization of Small Molecules toward a G-Quadruplex Structure Formed in the Promoter Region of c-MYC Oncogene.

Overexpression of c-MYC oncogene is associated with cancer pathology. Expression of c-MYC is regulated by the G-quadruplex structure formed in the G-rich segment of nuclease hypersensitive element (NHE III1), that is, “Pu27”, which is localized in the promoter region. Ligand-induced stabilization of the Pu27 structure has been identified as a novel target for cancer therapeutics. Here, we have explored the library of synthetic compounds against the predefined binding site of Pu27. Three compounds were selected based on the docking analyses; they were further scrutinized using all atom molecular dynamics simulations in an explicit water model. Simulated trajectories were scrutinized for conformational stability and ligand binding free energy estimation; essential dynamic behavior was determined using principal component analysis. One of the molecules, “TPP (1-(3-(4-(1,2,3-thiadiazol-4-yl)phenoxy)-2-hydroxypropyl)-4-carbamoylpiperidinium)”, with the best results was considered for further evaluation. The theoretical observations are supported well by biophysical analysis using circular dichroism, isothermal titration calorimetry, and high-resolution NMR spectroscopy indicating association of TPP with Pu27. The in vitro studies were then translated into c-MYC overexpression in the T47D breast cancer cell line. Biological evaluation through the MTT assay, flow cytometric assay, RT-PCR, and reporter luciferase assay suggests that TPP downregulates the expression of c-MYC oncogene by arresting its promoter region. In silico and in vitro observations cumulatively suggest that the novel skeleton of TPP could be a potential anticancer agent by stabilizing the G-quadruplex formed in the Pu27 and consequently downregulating the expression of c-MYC oncogene. Derivation of new molecules on its skeleton may confer anticancer therapeutics for the next generation.

Abstract 5230: Targeting two unique end-insertion G-quadruplexes formed in the 3′-end of the PDGFR-β core promoter nuclease hypersensitive element with ellipticine analog

Abstract Aberrant expression of PDGFR-β promotes multiple hallmarks of cancer, and its transcriptional regulation provides an attractive means to inhibit the PDGFR-β signaling pathway. The nuclease hypersensitive element (NHE) region of the human PDGFR-β core promoter has been shown to be responsible in regulating approximately 60% of the PDGFR-β basal promoter activity. Multiple overlapping G-quadruplexes (G4s) have been shown to form in this NHE region. We previously determined the most stable G4 structure formed in the 5’-mid of PDGFR-β NHE region. Significantly, an ellipticine analog, GSA1129, has been shown to selectively bind to the G4 formed in the 3’-end NHE region, thereby shifting the dynamic equilibrium toward the 3’-end G4. This selective interaction was shown to result in suppression of PDGFR-β activity in both cancer cell lines and a preclinical animal model. Thus, characterization of 3’-end G4 provides an important basis for understanding the molecular recognition of this G4 and for rational drug design. In this study, we used NMR spectroscopy to show that the PDGFR-β NHE 3′-end sequence forms two novel end-insertion intramolecular G4s which exist in a dynamic equilibrium under physiological salt condition. One is the 3′-insertion-G4 with a 3′ non-adjacent flanking guanine inserted into the 3′-external tetrad. The other is a 5′-insertion-G4 with a 5′ non-adjacent flanking guanine inserted into the 5′-external tetrad. We further confirmed that these novel end-insertion G4s can readily form in the 3’-end NHE sequence under physiologically relevant ionic conditions using dimethyl sulfate (DMS) footprinting experiments. To elucidate the molecular interactions of ellipticine analog with the 3’-end G4s, we studied the binding of GSA1129 to both of the end-insertion G4s. Significantly, our CD melting studies showed that GSA1129 markedly increased the stability of both end-insertion G4s by about 25°C. Furthermore, 1H NMR titration experiments have shown that GSA1129 selectively binds to the G4 formed in the 3’-end NHE region. The novel end-insertion structures and the dynamic nature of the PDGFR-β NHE 3′-end G4 may make it an effective target for GSA1129 binding and regulation. Our result highlights the dynamic nature of the PDGFR-β NHE 3′-end sequence and the importance of identifying the exact sequence for the formation of the naturally occurring G4 structure. Citation Format: Buket Onel, Prashansa Agrawal, Megan Carver, Laurence H. Hurley, Danzhou Yang. Targeting two unique end-insertion G-quadruplexes formed in the 3′-end of the PDGFR-β core promoter nuclease hypersensitive element with ellipticine analog [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5230. doi:10.1158/1538-7445.AM2017-5230

The Consequences of Overlapping G-Quadruplexes and i-Motifs in the Platelet-Derived Growth Factor Receptor β Core Promoter Nuclease Hypersensitive Element Can Explain the Unexpected Effects of Mutations and Provide Opportunities for Selective Targeting of Both Structures by Small Molecules To Downregulate Gene Expression.

The platelet-derived growth factor receptor β (PDGFR-β) signaling pathway is a validated and important target for the treatment of certain malignant and nonmalignant pathologies. We previously identified a G-quadruplex-forming nuclease hypersensitive element (NHE) in the human PDGFR-β promoter that putatively forms four overlapping G-quadruplexes. Therefore, we further investigated the structures and biological roles of the G-quadruplexes and i-motifs in the PDGFR-β NHE with the ultimate goal of demonstrating an alternate and effective strategy for molecularly targeting the PDGFR-β pathway. Significantly, we show that the primary G-quadruplex receptor for repression of PDGFR-β is the 3'-end G-quadruplex, which has a GGA sequence at the 3'-end. Mutation studies using luciferase reporter plasmids highlight a novel set of G-quadruplex point mutations, some of which seem to provide conflicting results on effects on gene expression, prompting further investigation into the effect of these mutations on the i-motif-forming strand. Herein we characterize the formation of an equilibrium between at least two different i-motifs from the cytosine-rich (C-rich) sequence of the PDGFR-β NHE. The apparently conflicting mutation results can be rationalized if we take into account the single base point mutation made in a critical cytosine run in the PDGFR-β NHE that dramatically affects the equilibrium of i-motifs formed from this sequence. We identified a group of ellipticines that targets the G-quadruplexes in the PDGFR-β promoter, and from this series of compounds, we selected the ellipticine analog GSA1129, which selectively targets the 3'-end G-quadruplex, to shift the dynamic equilibrium in the full-length sequence to favor this structure. We also identified a benzothiophene-2-carboxamide (NSC309874) as a PDGFR-β i-motif-interactive compound. In vitro, GSA1129 and NSC309874 downregulate PDGFR-β promoter activity and transcript in the neuroblastoma cell line SK-N-SH at subcytotoxic cell concentrations. GSA1129 also inhibits PDGFR-β-driven cell proliferation and migration. With an established preclinical murine model of acute lung injury, we demonstrate that GSA1129 attenuates endotoxin-mediated acute lung inflammation. Our studies underscore the importance of considering the effects of point mutations on structure formation from the G- and C-rich sequences and provide further evidence for the involvement of both strands and associated structures in the control of gene expression.

High-resolution three-dimensional NMR structure of the KRAS proto-oncogene promoter reveals key features of a G-quadruplex involved in transcriptional regulation

Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets, whose stacking leads to the formation of a G-quadruplex (G4). G4s can coexist with canonical duplex DNA in the human genome and have been suggested to suppress gene transcription, and much attention has therefore focused on studying G4s in promotor regions of disease-related genes. For example, the human KRAS proto-oncogene contains a nuclease-hypersensitive element located upstream of the major transcription start site. The KRAS nuclease-hypersensitive element (NHE) region contains a G-rich element (22RT; 5'-AGGGCGGTGTGGGAATAGGGAA-3') and encompasses a Myc-associated zinc finger-binding site that regulates KRAS transcription. The NEH region therefore has been proposed as a target for new drugs that control KRAS transcription, which requires detailed knowledge of the NHE structure. In this study, we report a high-resolution NMR structure of the G-rich element within the KRAS NHE. We found that the G-rich element forms a parallel structure with three G-quartets connected by a four-nucleotide loop and two short one-nucleotide double-chain reversal loops. In addition, a thymine bulge is found between G8 and G9. The loops of different lengths and the presence of a bulge between the G-quartets are structural elements that potentially can be targeted by small chemical ligands that would further stabilize the structure and interfere or block transcriptional regulators such as Myc-associated zinc finger from accessing their binding sites on the KRAS promoter. In conclusion, our work suggests a possible new route for the development of anticancer agents that could suppress KRAS expression.

Wild-type p53 binds to MYC promoter G-quadruplex.

G-quadruplexes are four-stranded nucleic acid structures that are implicated in the regulation of transcription, translation and replication. Genome regions enriched in putative G-quadruplex motifs include telomeres and gene promoters. Tumour suppressor p53 plays a critical role in regulatory pathways leading to cell cycle arrest, DNA repair and apoptosis. In addition to transcriptional regulation mediated via sequence-specific DNA binding, p53 can selectively bind various non-B DNA structures. In the present study, wild-type p53 (wtp53) binding to G-quadruplex formed by MYC promoter nuclease hypersensitive element (NHE) III1 region was investigated. Wtp53 binding to MYC G-quadruplex is comparable to interaction with specific p53 consensus sequence (p53CON). Apart from the full-length wtp53, its isolated C-terminal region (aa 320-393) as well, is capable of high-affinity MYC G-quadruplex binding, suggesting its critical role in this typeof interaction. Moreover, wtp53 binds to MYC promoter region containing putative G-quadruplex motif in two wtp53-expressing cell lines. The results suggest that wtp53 binding to G-quadruplexes can take part in transcriptional regulation of its target genes.

Discovery of a Family of Genomic Sequences Which Interact Specifically with the c-MYC Promoter to Regulate c-MYC Expression.

G-quadruplex forming sequences are particularly enriched in the promoter regions of eukaryotic genes, especially of oncogenes. One of the most well studied G-quadruplex forming sequences is located in the nuclease hypersensitive element (NHE) III1 of the c-MYC promoter region. The oncoprotein c-MYC regulates a large array of genes which play important roles in growth regulation and metabolism. It is dysregulated in >70% of human cancers. The silencer NHEIII1 located upstream of the P1 promoter regulates up-to 80% of c-MYC transcription and includes a G-quadruplex structure (Pu27) that is required for promoter inhibition. We have identified, for the first time, a family of seventeen G-quadruplex-forming motifs with >90% identity with Pu27, located on different chromosomes throughout the human genome, some found near or within genes involved in stem cell maintenance or neural cell development. Notably, all members of the Pu27 family interact specifically with NHEIII1 sequence, in vitro. Crosslinking studies demonstrate that Pu27 oligonucleotide binds specifically to the C-rich strand of the NHEIII1 resulting in the G-quadruplex structure stabilization. Pu27 homologous sequences (Pu27-HS) significantly inhibit leukemic cell lines proliferation in culture. Exposure of U937 cells to the Pu27-HS induces cell growth inhibition associated with cell cycle arrest that is most likely due to downregulation of c-MYC expression at the RNA and/or protein levels. Expression of SOX2, another gene containing a Pu27-HS, was affected by Pu27-HS treatment as well. Our data suggest that the oligonucleotides encoding the Pu27 family target complementary DNA sequences in the genome, including those of the c-MYC and SOX2 promoters. This effect is most likely cell type and cell growth condition dependent. The presence of genomic G-quadruplex-forming sequences homologous to Pu27 of c-MYC silencer and the fact that they interact specifically with the parent sequence suggest a common regulatory mechanism for genes whose promoters contain these sequences.

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

Abstract 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.

Abstract 4853: BMVC specifically binds the major G-quadruplex structure formed in the c-MYC promoter to lower c-MYC levels

Abstract The c-MYC proto-oncogene is one of the most deregulated genes in human cancers. Transcriptional repression of c-MYC is considered as one of the attractive strategies in targeting c-MYC. Previous studies revealed that the c-MYC promoter nuclease hypersensitive element (NHE) III1 region, which regulates 80-95% of total c-MYC transcription, can form DNA G-quadruplex (G4) and that stabilization of the c-MYC G4 could repress c-MYC gene expression. We have previously determined the molecular structure of the major G-quadruplex formed in the c-MYC promoter NHE (MycG4), which is an intramolecular parallel-stranded structure. BMVC, a carbazole derivative, was designed as a fluorescent probe for recognizing telomeric G4. However, we found that BMVC binds to MycG4 with much greater affinity and specificity. Cellular studies showed that BMVC was enriched in the nucleus at 48 hr after treatment, leading to markedly reduced c-MYC expression levels. Promoter-driven luciferase assays revealed that BMVC could enhance the inhibitory effects of MycG4, suggesting BMVC can repress c-MYC expression in vivo through stabilization of MycG4. To understand the molecular interactions between BMVC and MycG4, we have determined the molecular structure of the complexes of BMVC with MycG4 by NMR. BMVC appears to bind the 5’-end of the MycG4 with very high affinity, as shown by the slow-exchange binding on the NMR timescale. The structure of the 5’-end complex of BMVC and MycG4 shows that BMVC is paired to the flanking adenine with specific hydrogen bonding interactions, while the BMVC-adenine plane stacks nicely on the 5’-end G-tetrad of MycG4. Compared to the previous MycG4 complex with a quindoline compound, the tighter and more specific binding of BMVC appears to be imparted by the novel arched shape of BMVC and specific pairing recognition of BMVC with the flanking bases of MycG4. BMVC can also bind to the 3’-end of MycG4 with lower affinity to form a second complex. However, the 3’-end complex shows much more dynamic conformation and less specific interactions. Our results indicated that the cellular targets of BMVC include not only telomeric G4, but also the c-MYC promoter G4. Because of the inherent fluorescence and novel binding mode of BMVC, our study provides useful information for future design of improved carbazole-based anticancer agents or specific cellular fluorescent probes, as well as insights into specific recognition of MycG4 by small molecules. Citation Format: Wenting Liu, Guanhui Wu, Clement Lin, Buket Onel, Ding Chen, Ta-Chau Chang, Danzhou Yang. BMVC specifically binds the major G-quadruplex structure formed in the c-MYC promoter to lower c-MYC levels. [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 4853.

KRAS promoter oligonucleotide with decoy activity dimerizes into a unique topology consisting of two G-quadruplex units.

Mutations of the KRAS proto-oncogene are associated with several tumor types, which is why it is being considered as a target for anti-cancer drug development. The human KRAS promoter contains a nuclease hypersensitive element (NHE), which can bind to nuclear proteins and is believed to form G-quadruplex structures. Previous studies showed that a 32-nt oligonucleotide (32R-3n) mimicking the KRAS NHE can reduce gene transcription by sequestering MAZ, a crucial transcription factor. Here we show that 32R-3n has to dimerize in order to fold into a G-quadruplex structure. Individual 5′- and 3′-end G-quadruplex units are formed and both feature a symmetric head-to-head topology with edge-type loops. The MAZ binding sequence is located within the 3′-end unit. Nuclear magnetic resonance data complemented by CD and UV spectra show that nucleotides of the MAZ binding G-rich motif are dynamic and could be available for sequence or structure based recognition. Both stable G-quadruplex structures could protect 5′- and 3′-ends of 32R-3n and enhance its anti-cancer activity. Single stranded genomic KRAS NHE including nucleotides flanking the 32R-3n sequence could favor a different monomeric fold, which remains unknown.

Abstract 5418: Lipid modification of c-MYC promoter targeted oligonucleotide stabilizes G-quadruplex formation and enhances its growth inhibitory activity in leukemia cells

Abstract Deregulation c-MYC is considered to be the hallmark of cancer as it regulates a large array of key genes essential for cell function such as proliferation, metabolism, differentiation, adhesion and apoptosis. Consequently, c-MYC expression is tightly regulated at the transcriptional and post-transcriptional levels. One of the key regulators of c-MYC transcription is the Nuclease Hypersensitive Element III1 (NHEIII1) located up-stream of P1 promoter. NHEIII1 is a gene silencer requiring for its function a stable spatial conformation that is provided by the high G/C rich ratio that forms G-quadruplex and i-motif structures. The oligonucleotide sequence Pu27 (27 nucleotides) forms G-quadruplex and correspond to the genomic NHEIII1 sequence. We have shown that Pu27 reduces c-MYC transcription in leukemia cell lines and consequently inhibits cell growth and promotes apoptosis. In this study, we evaluated the effect of Pu27 modification using polyethylene glycol (PEG), tocopherol (Toco) and the lipid palmitate (Palmi) in order to increase G-quadruplex stability and lessen blood clearance. We investigated the binding property to NHEIII1 target sequence using electrophoretic mobility shift assay (EMSA). The target used for this study was a 134bp covering NHEIII1 as double stranded (ds) and C-rich or G-rich single stranded (ss) DNA. We verify the presence of G-quadruplex formation in the modified Pu27 using circular dichroism (CD) spectrometry in two different salinity conditions 100mM KCl: that favors G-quadruplex formation and 100mM NaCl that does not. In addition, the effect of the modified-Pu on cell proliferation was investigated in U937 and Raji leukemia cell lines using MTT assay. We show that Pu27 binds specifically to the target sequence on the C-rich strand. The CD spectra confirm the presence of G-quadruplex in the modified Pu27. Furthermore, the CD measurement in two different salinity conditions revealed that Toco and Palmi- modifications stabilize the formation of G-quadruplex even in less favorable condition (100mM NaCl). The dose response study in MTT assay reveals that Toco and Palmi modified-Pu inhibit cell growth for Raji and U937 leukemia cell lines as well, if not better than unmodified Pu27. This data demonstrates the specific binding of the G-quadruplex lipid-modified Pu27 at the silencer element in a sequence specific manner (by strand invasion) and down regulation of c-MYC expression as is the case for unmodified Pu27. Subsequently down regulation of c-MYC by Pu27 was followed by change in c-MYC target genes expression and inhibition of cell growth. Our finding suggests that modification of the c-MYC targeted oligonucleotide by addition of lipids stabilizes the 3D structure (G-quadruplex) and improve its function at inhibiting cell growth most likely by down-regulating c-MYC. Citation Format: Francine Francine, Shelia D. Thomas, Gilles Tapolsky, Donald M. Miller. Lipid modification of c-MYC promoter targeted oligonucleotide stabilizes G-quadruplex formation and enhances its growth inhibitory activity in leukemia cells. [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 5418. doi:10.1158/1538-7445.AM2015-5418

Abstract 1629: Identification and biological characterization of a novel class of small molecules to inhibit c-myc transcription

Abstract The transcription factor c-myc, deregulated in ∼70% of all cancer cases, facilitates tumor initiation and promotion most frequently in colon, breast, lung, ovarian, and lymphoid malignancies, and acts as a universal amplifier of aberrant growth-related cell signaling. Due to its helix-loop helix topology, short protein half-life, and rapid replenishment within the cancer cell, c-myc was long considered to be ‘undruggable.’ Recently, much attention has focused on inhibiting c-myc either through indirect pathway modulation, or on the transcriptional level. 85-90% of c-myc gene expression is controlled by a G-quadruplex (G4) DNA structure found in the nuclease hypersensitive element III(1) region (NHEIII1) of its promoter, which, when stabilized with a chemical agent, would theoretically inhibit myc transcription by preventing normal polymerase function. In this study, we report the identification and biological characterization of a novel chemical class to directly bind the c-myc G4 DNA. The chemical motif discussed here was identified from a highly selective (hit rate of 0.16%, with few false positives) small molecule microarray screen of 20,000 compounds. A panel of the 12 most promising hits was chosen for biological and biochemical secondary validation in a variety of in vitro and cell based assays to confirm mechanism of action and functional effects in biological systems. Of note, numerous compounds from the initial panel of hits exhibited effects with a range of potency in comparison with untreated and non quadruplex forming nucleic acid controls, thereby validating the original screening method. For prioritization, hits were further evaluated for their capacity to preferentially induce myc-dependent cell death in a tetracycline controlled c-myc stable cell line. A top lead was identified, and structure-activity relationship was preliminarily evaluated with a small panel of analogues, leading to a final compound with three times greater potency. The chosen compound exhibited strong dose-dependent cell killing activity, in human multiple myeloma with high levels of c-myc, with an IC50 in the single digit micromolar range. Additionally, the compound reduced c-myc gene expression in a series of B cell neoplasms to near the limit of detection, both transcriptionally and translationally. The mechanism of action was confirmed in a Burkitt's Lymphoma model through a quadruplex promoter-specific comparative analysis of myc transcript copy numbers in the presence or absence of treatment. Continuing studies will focus on further structural optimization of the ligand, a thorough examination of its genome-wide effects, and tolerability in animal models. Citation Format: Kenneth M. Felsenstein, John K. Simmons, Peter Gareiss, Beverly A. Mock, John ‘Jay’ S. Schneekloth. Identification and biological characterization of a novel class of small molecules to inhibit c-myc transcription. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1629. doi:10.1158/1538-7445.AM2014-1629

Synergic Role of Nucleophosmin Three-helix Bundle and a Flanking Unstructured Tail in the Interaction with G-quadruplex DNA

Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein, mainly localized at nucleoli, that plays a number of functions in ribosome biogenesis and export, cell cycle control, and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia; mutations map to the C-terminal domain of the protein and cause its denaturation and aberrant cytoplasmic translocation. NPM1 C-terminal domain binds G-quadruplex regions at ribosomal DNA and at gene promoters, including the well characterized sequence from the nuclease-hypersensitive element III region of the c-MYC promoter. These activities are lost by the leukemic variant. Here we analyze the NPM1/G-quadruplex interaction, focusing on residues belonging to both the NPM1 terminal three-helix bundle and a lysine-rich unstructured tail, which has been shown to be necessary for high affinity recognition. We performed extended site-directed mutagenesis and measured binding rate constants through surface plasmon resonance analysis. These data, supported by molecular dynamics simulations, suggest that the unstructured tail plays a double role in the reaction mechanism. On the one hand, it facilitates the formation of an encounter complex through long range electrostatic interactions; on the other hand, it directly contacts the G-quadruplex scaffold through multiple and transient electrostatic interactions, significantly enlarging the contact surface.