Parallel DNA G-quadruplexes (G4) are key regulators of oncogene transcription. The MYC promoter G-quadruplex (MycG4) is a prototype parallel G4 and promising anticancer drug target. However, effects of DNA damage or mutations on their conformational landscape remain elusive. Systematic analysis of mutational effects is challenging because permutating G4-forming sequences creates a vast sequence space inaccessible to most experimental methods. Herein, using a custom G4-DNA microarray, we systematically examine all 2 145 possible single and double mutations of the MYC promoter G4. The results show no single or even double mutation completely prevents MYC G-quadruplex formation, emphasizing its exceptional robustness. Mutated MycG4 sequences can form G-quadruplexes with vacancies or bulges without the need to replace the damaged G-runs. Both length and position of a G-run determine its resilience against mutations. Intriguingly, our results reveal an efficient compensation mechanism for mutations involving nearby redundant G-residues to preserve G4 formation. Moreover, the most disruptive mutations involve two nonadjacent G-runs, which cannot be repaired by a single G-tract but can still be compensated by redundant guanines. These results provide critical insights into G-quadruplex folding, structural resilience, and damage tolerance, with implications for gene regulation and G4-targeted drug design.

Overexpression of PDGFR-β (platelet-derived growth factor receptor beta) kinase contributes to diverse human diseases, including cancers, cardiovascular disorders, and fibrosis. G-quadruplexes (G4s) formed in the PDGFR-β promoter act as transcriptional repressors and represent attractive therapeutic targets. We previously reported that the major G4-forming region of the PDGFR-β promoter adopts a unique broken-strand G4, whereas truncation of this sequence generates a vacancy G4 (vG4) that can be filled-in by external guanine analogs or metabolites and further stabilized by small molecules, suggesting a potential regulatory mechanism and opportunity for selective drug targeting. However, the relationship between broken-strand G4s and vG4s remains unclear. Here, we demonstrate that the PDGFR-β promoter sequence forms a dynamic equilibrium between two broken-strand G4 conformations that interconvert on the millisecond timescale, with vG4 serving as an intermediate. We determined the high-resolution NMR structures of these interconverting G4s, which share a conserved vG4 core but differ in their intramolecular guanine "fill-in." Both conformations feature a stabilizing G-G capping base pair unique to the PDGFR-β promoter. These findings elucidate the structural details of broken-strand PDGFR-β promoter G4s and the mechanism of vG4 formation, providing critical insights for selective drug targeting and establishing a framework for rational design of small molecules to modulate PDGFR-β transcription.

Abstract Background: G-quadruplex (G4) DNA and RNA are important non-canonical nucleic acid secondary structures that play key roles in many cellular processes. They regulate the expression and translation of several oncogenes, including the master cell growth regulator, MYC. Bisantrene is a small-molecule anticancer agent that has been shown to be safe and effective in >1500 clinical trial patients. This study characterized the binding and stabilization of a G4 region in the c-MYC promoter by the (E,E)-bisantrene isomer and silencing of c-MYC expression in cancer cells. Methods: Circular dichroism spectroscopy established if (E,E)-bisantrene stabilizes the c-MYC promotor G4 structure, with surface plasmon resonance used to measure the binding affinity. Nuclear magnetic resonance spectroscopy provided structural insights into the binding interactions within the complex. Molecular dynamics simulations modelled the 3-dimensional structure of the complex. Changes in c-MYC gene expression were assessed across a range of cancer cell lines after treatment with (E,E)-bisantrene and RNA-seq with pathway analysis was performed. Results: (E,E)-bisantrene was found to stabilize the c-MYC promoter G4 region, producing similar increases in melting temperature to other G4 ligands (i.e., pidnarulex and pyridostatin). NMR spectroscopy and molecular modelling suggests (E,E)-bisantrene binds with a 2:1 stoichiometry to the planar surfaces of the top and bottom G-tetrads. (E,E)-bisantrene potently inhibited c-MYC expression in multiple cancer cell lines. RNA-seq analysis showed (E,E)-bisantrene also decreased expression of other oncogenes containing G4 regions in their promoters, including MET, TERT, VEGFA, ATF4 and MDM2. Pathway analysis of the RNA-Seq data demonstrated a transcriptomic profile similar to pidnarulex, a known G4-binding drug in early-stage clinical development. Conclusion: (E,E)-bisantrene binds to and stabilizes the G4 structure contained within the c-MYC promotor region, leading to silencing of c-MYC gene expression. These studies support clinical evaluation of (E,E)-bisantrene as a new G4-targeting drug in MYC-driven tumors. Citation Format: Sumit Sahni, Emily Ryan, Peter Cuthbertson, Feroz Ahmad, Kirsten Curnow, Nehad Elsalamouny, Qiang Zhu, Haibo Yu, Jinho Jang, Jonathan Dickerhoff, Danzhou Yang, Emma-Jayne Proctor, Martina Sanderson-Smith, Daniel Tillett, Michael Kelso. (E,E)-bisantrene silences c-MYC expression by stabilizing its promotor region G-quadruplex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5751.

Bloom syndrome protein (BLM), a RecQ family DNA helicase, is consistently overexpressed in multiple malignancies, yet its therapeutic potential remains largely unexplored. Herein, we focused on targeting the BLM promoter G-quadruplex (BLM-G4) to inhibit the BLM signaling pathway. We first characterized the parallel BLM-G4 in the BLM promoter region. Subsequently, it is shown for the first time that BLM-G4 recruits phosphorylated signal transducer and activator of transcription 1 (pSTAT1) to activate BLM expression. Importantly, two natural alkaloids, berberine (BER) and coptisine (COP), compete with STAT1 for binding to BLM-G4, thereby significantly suppressing BLM expression in colon cancer cells. The BER/COP-BLM-G4 complex structures were determined using nuclear magnetic resonance experiments, which provide valuable insights for the rational design of next-generation BLM-G4-targeting ligands. Beyond BLM regulation, the conjoint analysis of genome-wide STAT1-CUT&Tag-seq, G4-CUT&Tag-seq, and COP-RNA-seq demonstrated STAT1 as a general G4-binding transcription factor and COP as a pan-genomic G4 stabilizer. Furthermore, BER/COP exhibited a pronounced synergistic effect with olaparib in inducing colon cancer cell death by disrupting DNA repair pathways and intensifying DNA damage. Collectively, our findings reveal a novel epigenetic mechanism of BLM gene upregulation mediated by BLM-G4-STAT1 interaction and suggest that the combination therapy of G4 stabilizers with poly(ADP) ribose polymerase (PARP) inhibitors is a promising strategy for treating complex cancers.

G-quadruplexes (G4s) have emerged as one of the most exciting nucleic acid secondary structures. G-quadruplexes are non-canonical, four-stranded nucleic acid structures formed in sequences with consecutive runs of guanine bases. Unlike duplex DNA, G-quadruplexes are globularly folded structures and can readily form under physiologically relevant solution conditions. G-quadruplex structures have been found in biologically significant nucleic acid regions, including human telomeres, oncogene-promoter regions, replication initiation sites, and untranslated regions (UTRs) of mRNA. They have been shown to be important regulatory motifs in a number of critical cellular processes including gene transcription, translation, DNA replication, and genomic stability. G-quadruplexes have become a new class of molecular targets for drug development. Nuclear magnetic resonance (NMR) spectroscopy is the major method for studying the structures of G-quadruplexes under physiologically relevant solution conditions. NMR spectroscopy is a powerful tool for studying G-quadruplex interactions with small molecule ligands in solution. To date, most G-quadruplex structures have been determined using NMR techniques. This review provides a comprehensive overview of the NMR methods used to determine DNA G-quadruplex structures and their ligand interactions in solution. It covers essential steps such as resonance assignment, which is foundational for all NMR studies, as well as the determination of G-quadruplex folding topology and structure using NMR spectroscopy. Additionally, it discusses NMR structural studies of small molecule interactions with DNA G-quadruplexes. Through examples of NMR-based structure characterization of G-quadruplexes and G-quadruplex-ligand complexes, this review illustrates the rich information that NMR spectroscopy can provide, demonstrating its applicability to a broad range of biologically relevant DNA G-quadruplexes and their ligand interactions.

The MYC oncogene promoter G-quadruplex (MycG4) regulates transcription and is a prevalent G4 locus in immortal cells. Nucleolin, a major MycG4-binding protein, exhibits greater affinity for MycG4 than for nucleolin recognition element (NRE) RNA. Nucleolin's four RNA binding domains (RBDs) are essential for high-affinity MycG4 binding. We present the 2.6-angstrom crystal structure of the nucleolin-MycG4 complex, revealing a folded parallel three-tetrad G-quadruplex with two coordinating potassium ions (K+), interacting with RBD1, RBD2, and Linker12 through its 6-nucleotide (nt) central loop and 5' flanking region. RBD3 and RBD4 bind MycG4's 1-nt loops as demonstrated by nuclear magnetic resonance (NMR). Cleavage under targets and tagmentation sequencing confirmed nucleolin's binding to MycG4 in cells. Our results revealed a G4 conformation-based recognition by a regulating protein through multivalent interactions, suggesting that G4s are nucleolin's primary cellular substrates, indicating G4 epigenetic transcriptional regulation and helping G4-targeted drug discovery.

Innate immune responses rely on a critical adaptor protein, MYD88, to bridge extracellular inflammatory signals and transcription factor networks inside the cell. Dysregulation of MYD88 is associated with immunodeficiencies, autoimmunity, and cancer. Here, we identify a stretch of guanine/cytosine-rich DNA in the MYD88 promoter capable of adopting stable G-quadruplex and i-Motif structures. Molecular characterization of the i-motif reveals a unique folding pattern with asymmetric lateral loop sizes, a transition pH in line with previously documented i-motifs, and in vitro recognition by the chromatin insulator/transcription factor (CTCF). In exploring the transcriptional role and therapeutic potential of the MYD88 structures, we show the known G-quadruplex ligand, TMPyP4, destabilizes the i-motif, stabilizes the G-quadruplex, and promotes MYD88 expression. A ligand, 33353, from the National Cancer Institute (NCI) Diversity Set, also differentially interacts with the two structures yet represses MYD88. This work discovers DNA structures in MYD88 that can be pharmacologically leveraged for their ability to control gene expression.

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. 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.

G-quadruplex (G4) structures play integral roles in modulating biological functions and can be regulated by small molecules. The MYC gene is critical during tumor initiation and malignant progression, in which G4 acts as an important modulation motif. Herein, we reported the MYC promoter G4 recognized by a platinum(II) compound Pt-phen. Two Pt-phen-MYC G4 complex structures in 5 mM K+ were determined by NMR. The Pt-phen first strongly binds the 3'-end of MYC G4 to form a 1:1 3'-end binding complex and then binds 5'-end to form a 2:1 complex with more Pt-phen. In the complexes, the Pt-phen molecules are well-defined and stack over four bases at the G-tetrad for a highly extensive π-π interaction, with the Pt atom aligning with the center of the G-tetrad. The flanking residues were observed to rearrange and cover on top of Pt-phen to stabilize the whole complex. We further demonstrated that Pt-phen targets G4 DNA in living cells and represses MYC gene expression in cancer cells. Our work elucidated the structural basis of ligand binding to MYC promoter G4. The platinum compound bound G4 includes multiple complexes formation, providing insights into the design of metal ligands targeting oncogene G4 DNA.

NMR spectroscopy is the major method for G-quadruplex structure determination under physiologically relevant solution conditions. Unlike duplex B-DNA, in which all nucleotides adopt an anti glycosidic conformation, the core tetrad-guanines in a G-quadruplex can adopt anti or syn glycosidic conformation depending on the folding structure. An experimental method that can clearly and unambiguously determine syn and anti tetrad-Gs in a G-quadruplex is highly desirable and necessary. In the present study, we exploit the advantages of the 1H-13C HSQC experiment to determine tetrad-G's glycosidic conformation and thus folding topology of G-quadruplexes. We use several examples to demonstrate the clear and straightforward determination of the guanine glycosidic conformations and G-quadruplex folding structures. Moreover, 1H-13C HSQC data can readily identify adenine H2 resonances as well as determine unusual syn conformation in loop and flanking sequences, a challenging task by standard 2D NOESY.

Abstract Metastatic breast cancer (MBC) is the most advanced stage of breast cancer. Our understanding of the molecular mechanisms which drive MBC remain incomplete. Epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET) promote drug resistance and metastasis. It has been reported that fibroblast growth factor receptor 1 (FGFR1) plays a key role during the EMT:MET cycle. Furthermore, FGFR1 is amplified in 13% of primary and 20% of metastatic breast cancer patients. Therefore, optimizing inhibition of FGFR1 is crucial for the therapeutic targeting of the late stage breast cancer. First, we examined the efficacies of FGFR kinase inhibitors in the murine based dormant 4T07 tumor model. Inhibition of FGFR kinase activity leads to tumor growth inhibition but fail to eradicate dormant breast cancer cells. Therefore, we explored broader approaches to inhibit FGFR1 expression in addition to blockade of its kinase activity. G-quadruplex (G4) structures are secondary DNA structures commonly found upstream of transcriptional start sites (TSS) of oncogenes restricting their expression. Consequently, pharmacological stabilization of G4 structures within the promoters of cancer-related genes via use of small molecules has emerged as a promising therapeutic approach in cancer. Results herein demonstrate that the proximal promoter of FGFR1 contains sequences that form G4. Circular dichroism was used to verify formation of G4 in the FGFR1 proximal promoter. Importantly, use of the G4-binding compound CX-5461 stabilized the FGFR1 G4 structure, blocked the transcriptional activity of the FGFR1 proximal promoter and decreased FGFR1 expression. Therefore, we implemented the G4 stabilizers in FGFR1 expressing and metastatic drug-resistant BC cell lines. This approach results in dramatic downregulation of FGFR1 at the protein level after treatment with the G4 stabilizer. G4 stabilizing agents also interfere with ectopic FGFR1 expression and EMT-driven FGFR1 expression. Importantly, use of the G4-targeting compound CX5461 effectively blocked FGFR1 expression and inhibited FGFR1 downstream signaling, resulting in eradication of dormant breast cancer cells. Finally, in vivo application of CX5461 reduced FGFR1 expression, blocked pulmonary tumor formation and prolonged animal survival. In conclusion, consistent with the clinical observations our evaluation of FGFR kinase inhibitors validates the resistance to FGFR kinase inhibitors in MBC. Our findings indicate that targeting FGFR1 expression through G4 stabilization may be a potential strategy for MBC. Citation Format: Muhammad Safdar, Hang Lin, Sarah Dagher, Jonathan Dickerhoff, Mitchell Ayers, Luis Solorio, Danzhou Yang, Michael Wendt, Saeed Akhand. Targeting fibroblast growth factor receptor (FGFR1) expression through G-quadruplex stabilization inhibits metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-18-06.

Limiting cellular plasticity is of key importance for the therapeutic targeting of metastatic breast cancer (MBC). Fibroblast growth receptor (FGFR) is a critical molecule in cellular plasticity and potent inhibitors of FGFR enzymatic activity have been developed, but kinase independent functions for this receptor also contribute to MBC progression. Herein, we evaluated several FGFR inhibitors and find that while FGFR-targeted kinase inhibitors are effective at blocking ligand-induced cell growth, dormant cells persist eventually giving rise to MBC progression. To more broadly target FGFR and cellular plasticity, we examined the FGFR1 proximal promoter, and found several sequences with potential to form G-quadruplex secondary structures. Circular dichroism was used to verify formation of G-quadruplex in the FGFR1 proximal promoter. Importantly, use of the clinical G-quadruplex-stabilizing compound, CX-5461, stabilized the FGFR1 G-quadruplex structures, blocked the transcriptional activity of the FGFR1 proximal promoter, decreased FGFR1 expression, and resulted in potent inhibition of pulmonary tumor formation. Overall, our findings suggest G-quadruplex-targeted compounds could be a potential therapeutic strategy to limit the cellular plasticity of FGFR1 overexpressing MBC. This study demonstrates the regulation of FGFR1 expression in metastatic breast cancer through the stabilization of G-quadruplex in its proximal promoter.

G-quadruplex (G4) DNA can form highly stable secondary structures in the presence of metal cations, and research has shown its potential as a transcriptional regulator for oncogenes in the human genome. In order to explore the interactions of DNA with metal cations using mass spectrometry, employing complementary fragmentation methods can enhance structural information. This study explores the use of ion-ion reactions for sequential negative electron transfer collision-induced dissociation (nET-CID) as a complement to traditional ion-trap CID (IT-CID). The resulting nET-CID data for G4 anions with and without metal cations show an increase in fragment ion type diversity and yield of structurally informative ions relative to IT-CID. The nET-CID yields greater sequence coverage by virtue of fragmentation at the 3'-side of thymine residues, which is lacking with IT-CID. Potassium adductions to backbone fragments in IT-CID and nET-CID spectra were nearly identical. Of note is a prominent fragment resulting from a loss of a 149 Da anion seen in nET-CID of large, G-rich sequences, proposed to be radical anion guanine loss. Neutral loss of neutral guanine (151 Da) and deprotonated nucleobase loss (150 Da) have been previously reported, but this is the first report of radical anion guanine loss (149 Da). Confirmation of the identity of the 149 Da anion results from the examination of the homonucleobase sequence 5'-GGGGGGGG-3'. Loss of a charged adenine radical anion at much lower relative abundance was also noted for the sequence 5'-AAAAAAAA-3'. DFT modeling indicates that the loss of a nucleobase as a radical anion from odd-electron nucleic acid anions is a thermodynamically favorable fragmentation pathway for G.

DNA guanine (G)-quadruplexes (G4s) are unique secondary structures formed by two or more stacked G-tetrads in G-rich DNA sequences. These structures have been found to play a crucial role in highly transcribed genes, especially in cancer-related oncogenes, making them attractive targets for cancer therapeutics. Significantly, targeting oncogene promoter G4 structures has emerged as a promising strategy to address the challenge of undruggable and drug-resistant proteins, such as MYC, BCL2, KRAS, and EGFR. Natural products have long been an important source of drug discovery, particularly in the fields of cancer and infectious diseases. Noteworthy progress has recently been made in the discovery of naturally occurring DNA G4-targeting drugs. Numerous DNA G4s, such as MYC-G4, BCL2-G4, KRAS-G4, PDGFR-β-G4, VEGF-G4, and telomeric-G4, have been identified as potential targets of natural products, including berberine, telomestatin, quindoline, sanguinarine, isaindigotone, and many others. Herein, we summarize and evaluate recent advancements in natural and nature-derived DNA G4 binders, focusing on understanding the structural recognition of DNA G4s by small molecules derived from nature. We also discuss the challenges and opportunities associated with developing drugs that target DNA G4s.

G-quadruplexes are noncanonical four-stranded DNA secondary structures. MYC is a master oncogene and the G-quadruplex formed in the MYC promoter functions as a transcriptional silencer and can be stabilized by small molecules. We have previously revealed a novel mechanism of action for indenoisoquinoline anticancer drugs, dual-downregulation of MYC and inhibition of topoisomerase I. Herein, we report the design and synthesis of novel 7-aza-8,9-methylenedioxyindenoisoquinolines based on desirable substituents and π-π stacking interactions. These compounds stabilize the MYC promoter G-quadruplex, significantly lower MYC levels in cancer cells, and inhibit topoisomerase I. MYC targeting was demonstrated by differential activities in Raji vs CA-46 cells and cytotoxicity in MYC-dependent cell lines. Cytotoxicities in the NCI-60 panel of human cancer cell lines were investigated. Favorable pharmacokinetics were established, and in vivo anticancer activities were demonstrated in xenograft mouse models. Furthermore, favorable brain penetration, brain pharmacokinetics, and anticancer activity in an orthotopic glioblastoma mouse model were demonstrated.

Abstract Overexpression of the receptor tyrosine kinase PDGFR-β promotes cancer growth and metastasis. PDGFR-β is a validated cancer target but PDGFR-β kinase inhibitors suffer from lack of selectivity and side effects. G-quadruplexes are an exciting class of non-B DNA secondary structures with functional importance. The G-quadruplex formed in the PDGFR-β promoter is a transcription repressor and targetable by small molecules. Therefore, the PDGFR-β promoter G-quadruplex is an attractive molecular target for anticancer drugs. Structural information of the PDGFR-β promoter G-quadruplex is crucial for understanding its cellular function and rational drug design. Herein, we used nuclear magnetic resonance (NMR) spectroscopy and biophysical methods to determine the structure and dynamics of the major G-quadruplex formed in the human PDGFR-β promoter. Unlike canonical G-quadruplexes formed by four runs of three continuous guanines (G), the major PDGFR-β G-quadruplex adopts a novel broken-strand structure where a two-G segment is continued by a non-adjacent guanine. The broken-strand G-quadruplex has a vacancy-G-tetrad (vG4) which is filled-in intramolecularly by a distal guanine. Interestingly, we discovered that the major PDGFR-β G-quadruplex is a novel mixture of two equilibrating broken-strand G-quadruplexes. Both structures adopt overall parallel-stranded folding with all 1-nt chain-reversal loops. However, a different 3’-filled-in guanine is delivered by a lateral loop, which forms structurally similar hairpin with a unique capping G-G base-pair. The formation of the equilibrating broken-strand G-quadruplexes and unique capping structures are both specific to the PDGFR-β promoter sequence. Intriguingly, the observed dynamic equilibrium between the two co-existing structures with different intramolecular fill-in guanines indicates a novel vG4 intermediate, which could be filled-in by cellular guanine metabolites such as cGMP. Therefore, our study provides critical insights into how the dynamics of the major PDGFR-β G-quadruplex lead to vG4 formation and potential regulation of gene transcription by cellular guanine metabolites. The determined molecular structures of the novel broken-strand G-quadruplexes provide a structural basis for rational design of small molecules to specifically target the major PDGFR-β promoter G-quadruplex for cancer therapeutics. Citation Format: Yichen Han, Jonathan Dickerhoff, Danzhou Yang. Structure and dynamics of the major PDGFR-β oncogene promoter G-quadruplex and insights into its cellular regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5775.

Metastatic breast cancer (MBC) is the most advanced stage of breast cancer. Our understanding of the molecular mechanisms which drive MBC remain incomplete. Epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET) promote drug resistance and metastasis. It has been reported that fibroblast growth factor receptor 1 (FGFR1) plays a key role during the EMT:MET cycle. Furthermore, FGFR1 is amplified in 13% of primary and 20% of metastatic breast cancer patients. Therefore, optimizing inhibition of FGFR1 is crucial for the therapeutic targeting of the late stage breast cancer. First, we examined the efficacies of FGFR kinase inhibitors in the murine based dormant 4T07 tumor model. Inhibition of FGFR kinase activity leads to tumor growth inhibition but fail to eradicate dormant breast cancer cells. Therefore, we explored broader approaches to inhibit FGFR1 expression in addition to blockade of its kinase activity. G-quadruplex (G4) structures are secondary DNA structures commonly found upstream of transcriptional start sites (TSS) of oncogenes restricting their expression. Consequently, pharmacological stabilization of G4 structures within the promoters of cancer-related genes via use of small molecules has emerged as a promising therapeutic approach in cancer. Results herein demonstrate that the proximal promoter of FGFR1 contains sequences that form G4. Circular dichroism was used to verify formation of G4 in the FGFR1 proximal promoter. Importantly, use of the G4-binding compound CX-5461 stabilized the FGFR1 G4 structure, blocked the transcriptional activity of the FGFR1 proximal promoter and decreased FGFR1 expression. Therefore, we implemented the G4 stabilizers in FGFR1 expressing and metastatic drug-resistant cell lines. This approach results in dramatic downregulation of FGFR1 in the protein level after treatment with the G4 stabilizer. G4 stabilizing agents also interfere with constitutive FGFR1 and EMT-driven FGFR1 expression. Importantly, use of the G4-targeting compound CX5461 effectively blocked FGFR1 expression and inhibited FGFR1 downstream signaling, resulting in eradication of dormant breast cancer cells using a 3D culture model system. Finally, in vivo application of CX5461 reduced pulmonary tumor growth and prolonged animal survival in an FGFR1-driven model of metastasis. In conclusion, our evaluation of FGFR kinase inhibitors validates clinically observed resistance to this approach in MBC. Moreover, our findings suggest that G4 stabilization may be a potential therapeutic strategy for FGFR1 expressing MBC. Citation Format: Muhammad Hassan Safdar, Hang Lin, Sarah Dagher, Jonathan Dickerhoff, Danzhou Yang, Michael K. Wendt. Targeted down regulation of FGFR1 through G-quadruplex stabilization in metastatic breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5005.

G-quadruplexes are noncanonical four-stranded DNA and RNA secondary structures formed in guanine-rich sequences. DNA G-quadruplexes are found in specific human genomic locations of functional significance, such as telomeres, promoters, and replication initiation sites. They are involved in essential cellular processes, such as genome stability, gene transcription, and DNA replication. DNA G-quadruplexes readily form under physiologically relevant solution conditions. Their globular shape sets them apart from the thread-like double-helix DNA. As such, DNA G-quadruplexes are considered a new class of molecular targets for drug development. Additionally, there is considerable interest in the use of G-quadruplexes for biomaterials, biosensors, and biocatalysts. Therefore, DNA G-quadruplex has emerged as one of the most exciting nucleic acid secondary structures. Structural information of DNA G-quadruplexes is essential to understand their biological functions, therapeutic interventions, and biomaterial applications. This chapter discusses structural characteristics of DNA G-quadruplexes and their drug interactions.