Robert V. Brown

Demonstration that Drug-targeted Down-regulation of MYC in Non-Hodgkins Lymphoma Is Directly Mediated through the Promoter G-quadruplex

Most transcription of the MYC proto-oncogene initiates in the near upstream promoter, within which lies the nuclease hypersensitive element (NHE) III1 region containing the CT-element. This dynamic stretch of DNA can form at least three different topologies: single-stranded DNA, double-stranded DNA, or higher order secondary structures that silence transcription. In the current report, we identify the ellipticine analog GQC-05 (NSC338258) as a high affinity, potent, and selective stabilizer of the MYC G-quadruplex (G4). In cells, GQC-05 induced cytotoxicity with corresponding decreased MYC mRNA and altered protein binding to the NHE III1 region, in agreement with a G4 stabilizing compound. We further describe a unique feature of the Burkitts lymphoma cell line CA46 that allowed us to clearly demonstrate the mechanism and location of action of GQC-05 within this region of DNA and through the G4. Most importantly, these data present, as far as we are aware, the most direct evidence of intracellular G4-mediated control of a particular promoter.

The major G-quadruplex formed in the human platelet-derived growth factor receptor β promoter adopts a novel broken-strand structure in K+ solution.

Overexpression of platelet-derived growth factor receptor β (PDGFR-β) has been associated with cancers and vascular and fibrotic disorders. PDGFR-β has become an attractive target for the treatment of cancers and fibrotic disorders. DNA G-quadruplexes formed in the GC-rich nuclease hypersensitivity element of the human PDGFR-β gene promoter have been found to inhibit PDGFR-β transcriptional activity. Here we determined the major G-quadruplex formed in the PDGFR-β promoter. Instead of using four continuous runs with three or more guanines, this G-quadruplex adopts a novel folding with a broken G-strand to form a primarily parallel-stranded intramolecular structure with three 1 nucleotide (nt) double-chain-reversal loops and one additional lateral loop. The novel folding of the PDGFR-β promoter G-quadruplex emphasizes the robustness of parallel-stranded structural motifs with a 1 nt loop. Considering recent progress on G-quadruplexes formed in gene-promoter sequences, we suggest the 1 nt looped G(i)NG(j) motif may have been evolutionarily selected to serve as a stable foundation upon which the promoter G-quadruplexes can build. The novel folding of the PDGFR-β promoter G-quadruplex may be attractive for small-molecule drugs that specifically target this secondary structure and modulate PDGFR-β gene expression.

Therapeutic Melting Pot of Never in Mitosis Gene A Related Kinase 2 (Nek2): A Perspective on Nek2 as an Oncology Target and Recent Advancements in Nek2 Small Molecule Inhibition

The global incidence of cancer is on the rise, and within the next decade, the disease is expected to become the leading cause of death worldwide. Forthcoming strategies used to treat cancers focus on the design and implementation of multidrug therapies to target complementary cancer specific pathways. A more direct means by which this multitargeted approach can be achieved is by identifying and targeting interpathway regulatory factors. Recent advances in understanding Nek2 (NIMA related kinase 2) biology suggest that the kinase potentially represents a multifaceted therapeutic target. In this regard, pharmacologic modulation of Nek2 with a single agent may effect several mechanisms important for tumor growth, survival, progression, and metastasis. We herein review the development of Nek2 as an oncology target and provide a succinct chronology of drug discovery campaigns focused on targeting Nek2.

DNA acting like RNA

Over the last decade or so, secondary non-B-DNA structures such as G-quadruplexes and i-motifs have come into focus as biologically functioning moieties that are potentially involved in telomeric interactions and the control of gene expression. In the present short review, we first describe the structural and dynamic parallels with complex RNA structures, including the importance of sequence and ions in folding, and then we describe the biological consequences of the folded structures. We conclude that there are considerable parallels between secondary and tertiary structures in RNA and DNA from both the folding and the biological perspectives.

Helping Eve Overcome ADAM: G-Quadruplexes in the ADAM-15 Promoter as New Molecular Targets for Breast Cancer Therapeutics

ADAM-15, with known zymogen, secretase, and disintegrin activities, is a catalytically active member of the ADAM family normally expressed in early embryonic development and aberrantly expressed in various cancers, including breast, prostate and lung. ADAM-15 promotes extracellular shedding of E-cadherin, a soluble ligand for the HER2/neu receptor, leading to activation, increased motility, and proliferation. Targeted downregulation of both ADAM-15 and HER2/neu function synergistically kills breast cancer cells, but to date there are no therapeutic options for decreasing ADAM-15 function or expression. In this vein, we have examined a unique string of guanine-rich DNA within the critical core promoter of ADAM-15. This region of DNA consists of seven contiguous runs of three or more consecutive guanines, which, under superhelical stress, can relax from duplex DNA to form an intrastrand secondary G-quadruplex (G4) structure. Using biophysical and biological techniques, we have examined the G4 formation within the entire and various truncated regions of the ADAM-15 promoter, and demonstrate strong intrastrand G4 formation serving to function as a biological silencer element. Characterization of the predominant G4 species formed within the ADAM-15 promoter will allow for specific drug targeting and stabilization, and the further development of novel, targeted therapeutics.

Abstract 4422: Direct demonstration of c-MYC G-quadruplex stabilization in vitro: elucidating the CA46 system

Many cancers harbor reciprocal chromosomal translocations that can lead to the activation of proto-oncogenes, inactivation of tumor-suppressor genes and/or generation of oncogenic chimera. In Non-Hodgkin Burkitt9s lymphoma (BL) there is a characteristic translocation of the c-MYC gene on chromosome 8q24 to one of three immunoglobulin genes on chromosomes 14, 2, or 22. The resulting product leads to c-MYC upregulation. ∼90% of c-MYC gene transcription is initiated by the P1 and P2 promoters, just upstream of which lies the guanine-rich NHE III1 control element. This is a very dynamic stretch of DNA, capable of forming at least three different DNA topologies: single stranded, double stranded and the G-quadruplex (G4) DNA. The G4 is a transcriptional silencer, and thus an attractive target for anti-cancer therapeutics. Using two BL cell lines (RAJI and CA46) we directly demonstrate the formation of a G4 in the c-MYC promoter. Using a small molecule compound, we clearly show specific activity dependent on the existence and regulation of this structure. The BL reciprocal translocation t(8;14) maintains G4-mediated control of c-MYC on both chromosomes in RAJI, but only on the non-translocated (NT) chromosome in CA46. The translocated (T) chromosome9s rate of transcription is 1000-fold greater than the NT, making the T the major allele, and the NT the minor allele. Thus, RAJI harbors G4-mediated control of both alleles and is expected to be more sensitive to G4-interactive compounds than CA46, which lost G4 control on the major allele. Indeed, GQC-05 (NSC338258), previously demonstrated to bind and stabilize the c-MYC G4, had greater cytotoxicity in the RAJI cell line. This compound demonstrated a rapid and time-dependent downregulation of c-MYC mRNA in RAJI, but no overt c-MYC effect in CA46. We were able to distinguish, using primers specific to exons 1 or 2, between the NT and T mRNA products, respectively. Remarkably, GQC-05 rapidly and significantly downregulated c-MYC mRNA from the NT allele in CA46 cells, where the G4 maintains transcriptional control. As a negative control, we examined the effects of GQC-05 on both exons in RAJI cells; no differential effect was observed. Doxorubicin is a non-G4 interactive compound that also decreases c-MYC expression in CA46 cells, but does not show this ‘exon-specific’ effect. Chromatin immunoprecipitation confirmed GQC-05 changes protein binding to the G4-region of the c-MYC promoter, with decreased transcription factor, and altered G4-regulatory factor, binding. Our work elucidates the primary mechanism of action for GQC-05 as stabilization of the c-MYC G4, leading to transcriptional downregulation. More importantly, we unambiguously demonstrate the formation and regulation of the c-MYC G4 in vitro and for the first time are able to evaluate c-MYC G4-targeted compounds in a whole cell system with this newly described CA46 model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4422. doi:10.1158/1538-7445.AM2011-4422

Abstract 1386: A novel approach to develop anti-VEGFR2/KDR therapeutics: characterizing and targeting the G-quadruplex

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The anti-angiogenic approach is traditionally considered for solid malignancies, but increasing evidence has also highlighted its involvement in the progression of hematological oncologies. Angiogenesis, including expression of key proteins, is aberrant in blood cancers such as leukemias, lymphomas, and myelomas, in addition to many solid malignancies, including pancreatic cancer. The most critical mediator of oncogenic angiogenesis is vascular endothelial growth factor (VEGF), which is recognized by two high-affinity receptor tyrosine kinases, VEGFR1 and VEGFR2 (KDR gene). VEGFR2 is necessary for the survival, growth, and differentiation of endothelial cells as its downstream effects include activation of the MAPK and the PI3K pathways, and its upregulation is observed under conditions of pathological angiogenesis. While VEGF/VEGFR2 signaling is known to act in a paracrine manner on endothelial cells, autocrine signaling has been observed in both malignant cells and hematopoietic stem cells. For endothelial and hematological malignancies, both autocrine and paracrine pathways can be targeted with anti-VEGFR2 therapies, potentially doubling the clinical efficacy. Intriguingly, the promoter region of KDR contains a GC-rich region of DNA within its the core promoter located −120 to −31 basepairs (bp) upstream of the transcriptional start site (TSS), putatively able to form several G-quadruplexes. These are unique DNA secondary structures that often serve as transcriptional silencer elements. In the present study, G-quadruplex formation has been isolated −80 to −38 bp upstream of the TSS, which contains five strings of guanines that form a mixed parallel/anti-parallel G-quadruplex, as determined by circular dichroism. The cleavage pattern of DMS footprinting and the major stop product of the polymerase stop assay confirmed that the dominant isoform is a parallel G-quadruplex occurring within the four 3’ runs of guanines. Mutant studies were performed to further determine guanines integral to G-quadruplex formation. An electrophoretic mobility shift assay was used to confirm the formation of a biologically relevant intramolecular structure. High-throughput screening identified a novel KDR-interactive agent, NSC643735, which thermally stabilized the G-quadruplex. This compound is currently being tested in vitro, and has high potential to transcriptionally downregulate KDR mRNA and, subsequently, VEGFR2 protein. These data characterizing the formation of a unique secondary structure in an important oncogene are promising to develop novel and specific anti-KDR therapy for the treatment of hematological and solid malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1386.

Abstract 3681: Helping Eve overcome ADAM: Characterization of the G-quadruplex in the ADAM15 core promoter

Breast cancer is the most common form of cancer in women, whose lethality is only surpassed by lung cancer. Through the last quarter-century there have been significant advances in detection and treatment options, such as Herceptin for HER2/neu positive cancers. While the development of targeted biological agents has greatly improved patient outcome, up to 70% of patients remain de novo refractory, relapse on therapy, or develop resistance. There is a pressing and eminent need for novel therapies to synergize with, or resensitize to, standard treatments. Enter ADAM15, with known zymogen, secretase, and disintegrin activies. This catalytically active member of the ADAM family is normally expressed in early embryonic development and is aberrantly expressed in various cancers, including breast. ADAM15 promotes extracellular shedding of E-cadherin, a soluble ligand for the HER2/neu receptor, leading to activation, increased motility, and proliferation. Seven independent microarray studies have shown that ADAM15 and HER2/neu are simultaneously upregulated in several stages of breast cancer, where their overexpression correlates with more aggressive and invasive disease. We have examined a unique string of GC-rich DNA within the critical core promoter of ADAM15. This region of DNA consists of seven tandem runs of three or more consecutive guanines. Under superhelical stress produced by the transcriptional complex, this region can relax from duplex DNA to form an intrastrand secondary structure known as a G-quadruplex. These globular entities generally serve as silencing elements for gene transcription. Furthermore, the topology of each G-quadruplex is as unique as a fingerprint. This allows for specific therapeutic targeting, offering a unique opportunity for treatment and selectivity over normal cells thus providing a potentially wide therapeutic window. Circular dichroism (CD) studies have confirmed the formation of a mixed parallel/anti-parallel G-quadruplex. Further CD analysis of the minimal G-quadruplex forming fragments has shown the capability of forming four unique thermally stable species. Electrophoretic mobility shift assay indicates the strongest intramolecular G-quadruplex formation in the 5′- and 3′-end runs of guanine, with minor intermolecular formations in the 3′-mid sequence, and a relatively unstable 5′-mid formation. To specifically determine which guanines are involved in the formation of the G-quadruplex and predict biologically relevant three-dimensional structures, DMS footprinting was performed on the full-length sequence and each of the four dissected regions. The pursuant data was used to build a molecular model of the structure. Full characterization of the G-quadruplex species formed will allow for specific drug targeting and stabilization, and the further development of novel, targeted therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3681.