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The Major G-Quadruplex Formed in the Human BCL-2 Proximal Promoter Adopts a Parallel Structure with a 13-nt Loop in K+ Solution
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The Major G-Quadruplex Formed in the Human BCL-2 Proximal Promoter Adopts a Parallel Structure with a 13-nt Loop in K+ Solution
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‡ § ∥ † ‡Department of Pharmacology and Toxicology, College of Pharmacy, †Department of Chemistry, §BIO5 Institute, ∥The Arizona Cancer Center, University of Arizona, 1703 East Mabel Street, Tucson, Arizona 85721, United States
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Cite this: J. Am. Chem. Soc. 2014, 136, 5, 1750–1753
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https://doi.org/10.1021/ja4118945
Published January 22, 2014

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Abstract

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The human BCL-2 gene contains a 39-bp GC-rich region upstream of the P1 promoter that has been shown to be critically involved in the regulation of BCL-2 gene expression. Inhibition of BCL-2 expression can decrease cellular proliferation and enhance the efficacy of chemotherapy. Here we report the major G-quadruplex formed in the Pu39 G-rich strand in this BCL-2 promoter region. The 1245G4 quadruplex adopts a parallel structure with one 13-nt and two 1-nt chain-reversal loops. The 1245G4 quadruplex involves four nonsuccessive G-runs, I, II, IV, V, unlike the previously reported bcl2 MidG4 quadruplex formed on the central four G-runs. The parallel 1245G4 quadruplex with the 13-nt loop, unexpectedly, appears to be more stable than the mixed parallel/antiparallel MidG4. Parallel-stranded structures with two 1-nt loops and one variable-length middle loop are found to be prevalent in the promoter G-quadruplexes; the variable middle loop is suggested to determine the specific overall structure and potential ligand recognition site. A limit of 7 nt in loop length is used in all quadruplex-predicting software. Thus, the formation and high stability of the 1245G4 quadruplex with a 13-nt loop is significant. The presence of two distinct interchangeable G-quadruplexes in the overlapping region of the BCL-2 promoter is intriguing, suggesting a novel mechanism for gene transcriptional regulation and ligand modulation.

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The BCL-2 (B-cell CLL/lymphoma 2) gene product is a mitochondrial membrane protein that plays an essential role in cell survival; it functions as an inhibitor of programmed cell death, or apoptosis. (1-3) BCL-2 has been found to be aberrantly overexpressed in a wide range of human tumors. (4-9) Elevation of BCL-2 levels has also been associated with poor prognosis and has been found to interfere with the traditional cancer therapeutics. (10, 11) Inhibition of BCL-2 expression by small molecules, (12, 13) peptidomimetics, (14) or antisense oligonucleotides (15, 16) has been shown to reduce cellular proliferation and to enhance chemotherapy efficacy. It has been shown that gene amplification or translocation can be equally common mechanisms causing BCL-2 overexpression in human cancer cells; (17) thus, effective modulation of BCL-2 expression offers promise for cancer therapeutics.

The human BCL-2 gene has two promoters, P1 and P2. The major promoter, P1, is located 1386–1423 base pairs upstream of the translation start site. (18) This is a TATA-less, GC-rich promoter that contains multiple transcriptional start sites and is positioned within a nuclease hypersensitive site. The 5′-end of the P1 promoter has been implicated in playing a major role in the regulation of BCL-2 transcription, (19) including a 39-bp GC-rich element that is located 57–19 base pairs upstream of the P1 promoter. Multiple transcription factors have been reported to bind to or regulate BCL-2 gene expression through this region, such as Sp1, (18) WT1, (20) E2F, (21) and NGF. (22) We have shown that the guanine-rich strand of the DNA in this region can form G-quadruplex structures, which can be stabilized by G-quadruplex-interactive agents. (23, 24) A second G-quadruplex-forming sequence has been found in the BCL-2 P1 promoter region; however, its stability appears to be much lower. (25) The 39-mer G-rich strand of this GC-rich element in the BCL-2 promoter region contains six runs of 3–5 consecutive guanines (Pu 39, Figure 1A) and has the potential to form 15 intramolecular G-quadruplex structures, using different combinations of four G-tracts. Our previous studies have shown that, on the three segments comprising the four successive runs of guanines of Pu39, i.e., I–IV (1234), II–V (2345), and III–VI (3456), the G-quadruplex formed on the central four G-runs (MidG4) appears to be the most stable in K+. (24) We have determined the folding pattern and molecular structure of MidG4, which forms a three-tetrad mixed parallel/antiparallel G-quadruplex with three loops of 1, 7, and 3 nt. (23, 26)

Figure 1

Figure 1. (A) The promoter sequence of the BCL-2 gene and its modifications. The top sequence is the 39-mer wild-type G-rich sequence (Pu39). The six G-runs with three or more guanines are underlined and numbered. Pu30 is the wild-type 30-mer G-rich sequence containing the I–V G-runs; the numbering used in this study is shown for Pu30. The guanine residues that are involved in the tetrad formation of the major BCL-2 G-quadruplex 1245G4 are shown in red. The mutations are shown in cyan. (B) DMS footprinting of the wild-type Pu39 with densitometric scans (left) and Pu30 (right). (C) Imino regions of 1D 1H NMR spectra of BCL-2 promoter sequences at 25 °C in 45 mM K+, pH 7.0. (D) CD spectra of Pu30 sequences in 95 mM K+.

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However, when we carried out dimethyl sulfate (DMS) footprinting on the full-length Pu39 in the presence of K+, we found that the G-run III, which is required for the MidG4 structure, is cleaved by DMS (Figure 1B left). In contrast, the G-runs I, II, IV, V are clearly protected from DMS cleavage (Figure 1B left). DMS footprinting can probe the formation of G-quadruplexes as the guanine residues involved in the G-tetrad formation are protected from the DMS cleavage. (27) Thus, it is indicated that the major G-quadruplex formed in K+ in the full-length Pu39 involves the four nonsuccessive G-runs, i.e., I, II, IV, and V, which we named 1245G4. We then carried out DMS footprinting of the wild-type Pu30 sequence comprising the 5′ five G-runs. The results showed that, consistent with the full-length Pu39 data, the major G-quadruplex formed in Pu30 did not involve the G-run III for the tetrad formation, or the 5′ two guanines of the G-run IV (Figure 1B right). Therefore, we prepared Pu30 sequences with modifications at G-runs III and IV (Figure 1A), including Pu30_3T with G-to-T mutations at G-run III, Pu30_4AA with G-to-A mutations at the first two guanines (G21 and G22) of G-run IV, and Pu30_3T4AA with mutations at both G-runs III and IV. The 1D 1H NMR spectra of the Pu30 sequences in K+ solution are shown in Figure 1C. Importantly, the imino proton regions at 10–12 ppm, characteristic of the formation of G-quadruplex structures, (26, 28) were very similar for all Pu30 sequences (Figure 1C), indicating that the same G-quadruplex is formed, and that G-run III and G21 and G22 of G-run IV were not involved in the tetrad formation. We have also carried out CD study of the wild-type and mutant Pu30 sequences, which were almost identical (Figure 1D). Therefore, the NMR and CD results were in good agreement with the DMS footprinting data (Figure 1B), that the G-run III and first two guanines of G-run IV are not involved in the G-tetrad formation of the major G-quadruplex (1245G4) formed in the BCL-2 promoter. We have also recorded 1D 1H NMR spectra of Pu39 and Pu30 (Figure S1), which showed that the imino protons of Pu30 could be detected in the major well-defined conformation in Pu39, although the NMR spectrum of Pu39 is not as well-resolved and appears to have higher-order structures co-present.

Pu30_3T4AA showed the best NMR spectral quality (Figure 1C), and was chosen for NMR structural analysis. For other sequences, however, the well-resolved spectrum can only be observed for the fresh sample at low DNA concentration; at higher concentration or with longer time, higher-order structures appeared to form as indicated by clearly elevated baseline (Figure S2). We prepared Pu30_3T4AA oligonucleotides with 6% incorporation of 15N-labeled guanine at each guanine position. The H1 proton of guanine is one-bond connected to N1, the guanine H8 proton is two-bond connected to N7 (Figure 2A); they are both readily detected for the site-specific labeled guanine by 1D 15N-edited NMR experiments. With such experiments, the H1 protons (Figure 2B left) and H8 protons (Figure 2B right) of each guanine were unambiguously assigned. Pu30_3T4AA contains 16 guanines (Figure 1A); only 12 imino peaks are observed in the 10.5–12 ppm region (Figure 2B left), indicating the formation of a three-tetrad G-quadruplex. G2 of G-run I, G11, G17, and G18 are not involved in the tetrad formation, consistent with the DMS footprinting data (Figure 1B right).

Figure 2

Figure 2. (A) A G-tetrad with detectable H1–H1 and H1–H8 NOE connectivity. (B) Imino H1 and aromatic H8 proton assignments of Pu30_3T4AA by 1D 15N-filtered experiments using site-specific labeled oligonucleotides at 25 °C. (C) Schematic drawing of the folding topology of the major G-quadruplex 1245G4 formed in the BCL-2 promoter sequence Pu30 (G = red, A = green, C = yellow). (D) H1–H1 region and (E) H1–H8 region of the 2D-NOESY spectrum of Pu30_3T4AA in H2O at 5 °C with a mixing time of 200 ms. The proton assignments are shown on the sides. Intratetrad NOEs are in red, intertetrad NOEs in blue, sequential intertetrad NOEs in black, and NOEs with flanking bases in green. Conditions: 25 mM K-phosphate, 70 mM KCl, pH 7.0.

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The assignment of the imino H1 and base H8 protons of guanines led to the direct determination of the folding topology of 1245G4 in the BCL-2 promoter (Figure 2C). A G-tetrad plane is connected with Hoogsteen H-bonds; the imino H1 protons of adjacent guanines, as well as the imino H1 and one adjacent guanine H8 protons, are in close spatial vicinity and detectable by NOE connections (Figure 2A). The arrangement and topology of a G-tetrad plane can thus be determined by guanine H1–H1 and H1–H8 NOEs (Figure 2C). For example, the NOE interactions between G3H1/G7H1, G7H1/G23H1, G23H1/G27H1, and G27H1/G3H1 (Figure 2D), and G7H8/G3H1, G23H8/G7H1, G27H8/G23H1, and G3H8/G27H1 (Figure 2E) defined a G-tetrad plane of G3-G7-G23-G27 (Figure 2C). The other two G-tetrad planes, i.e., G4-G8-G24-G28 and G5-G9-G25-G29 (Figure 2C), can be similarly defined. The G-quadruplex folding is further supported by intertetrad NOEs. These NOEs, such as G3H8/G28H1, G7H8/G4H1, G23H8/G8H1, and G27H8/G24H1 (Figure 2E), connect the top and middle G-tetrad planes (Figure 2C), while G4H8/G29H1, G8H8/G5H1, G24H8/G9H1, and G28H8/G25H1 (Figure 2E) connect the middle and bottom planes (Figure 2C), and reflect the right-handed twist of the DNA backbone. Our NMR results thus showed that the 1245G4 of the BCL-2 promoter is a 3-tetrad, parallel-stranded, intramolecular G-quadruplex structure, with three chain-reversal loops of 1, 13, and 1 nt, respectively (Figure 2C). All the tetrad-guanines are in anti glycosidic configuration, as shown by the medium intensities of intraresidue H8–H1′ NOEs (Figure S3). The CD spectra of Pu30 sequences showed a positive maximum at 264 nm and a negative minimum at 240 nm (Figure 1D), which are characteristic of parallel G-quadruplex structures, (29) supporting the parallel-stranded folding of the 1245G4. The melting temperature of Pu30_3T4AA was determined to be 71 °C as measured by the CD melting experiments at 264 nm (Figure S4) and NMR variable temperature study (Figure S5). The G-quadruplex formed by Pu30_3T4AA appeared to be of unimolecular nature as indicated by the concentration-independent melting temperature measured by CD and NMR.

The presence of a 13-nt loop in the major G-quadruplex 1245G4 formed in the BCL-2 promoter is unexpected and remarkable. DNA G-quadruplex secondary structures have been found to be overpresented in the human gene promoter regions as transcriptional regulators and are considered as a novel class of molecular targets for cancer therapeutics. (30) Intriguingly, parallel-stranded structures are found to be prevalent in the promoter G-quadruplexes, including those of c-MYC, (31-33) VEGF, (28) c-KIT21, (34) HIF-1α, (35) RET, (36) and hTERT (37, 38) (Figure 3). It has been shown that the chain-reversal loops in parallel G-quadruplexes greatly favor the short loop lengths, such as the G3NG3 motif with 1-nt loop. (39-42) The G3NG3 forms a robust parallel-stranded structural motif with 1-nt loop, which was first shown in the major c-MYC promoter quadruplex structure, (31) and is present in all other loop isomers formed in the c-MYC promoter. (29, 43) Indeed, most parallel-stranded promoter G-quadruplexes contain three tetrads and three chain-reversal loops, including two 1-nt loops and a variable-length middle loop (Figure 3). In the major c-MYC promoter G-quadruplex, a parallel structure with two 1-nt loops and one 2-nt middle loop, the 2-nt middle loop stays entirely in the groove. (31) In our recent study of the major human VEGF promoter G-quadruplex, a parallel structure with two 1-nt loops and a 4-nt middle loop (Figure 3), the 4-nt middle loop was found to stretch over the 5′ tetrad forming a unique capping structure with the 5′ flanking segment. (28) It is thus suggested that each parallel G-quadruplex adopts unique capping and loop structures by its specific middle loop and flanking segments, which likely determine the specific recognition sites of proteins or small molecules. By having two 1-nt loops, it appears that a stable parallel G-quadruplex can contain a more extended middle loop. However, the longest loop that has been reported to-date in a parallel G-quadruplex structure formed on naturally occurring sequences is 6 nt long. (33) In fact, in all the available G-quadruplex-predicting software, a limit of 7 nt is used for loop lengths. (44, 45) The formation and high stability of the 1245G4 G-quadruplex with a 13-nt chain-reversal loop in the BCL-2 promoter sequence is thus significant and would expand the current knowledge of DNA G-quadruplexes.

Figure 3

Figure 3. G-quadruplex-forming promoter sequences.

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It is highly intriguing that the two stable intramolecular structures formed in the BCL-2 promoter, i.e., 1245G4 and MidG4, adopt completely different folding structures. The presence of two distinct interchangeable G-quadruplexes in the overlapping region of the BCL-2 promoter could be important for the precise regulation of gene transcription, as each G-quadruplex is likely to be recognized by different proteins leading to different gene modulation. While thermodynamically the 1245G4 quadruplex is slightly more stable than MidG4 (Tm of 66 °C), the MidG4 quadruplex could be kinetically more favored due to its shorter loop-lengths. In addition, the two interchangeable G-quadruplexes may be recognized by different small molecules and proteins, which could differentially modulate BCL-2 gene transcription.

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Materials and methods, 1D 1H NMR spectra of Pu39 and Pu30 sequences, and H8/H6–H1′ region of the nonexchangeable 2D-NOESY, CD melting and NMR VT studies, of Pu30_3T4AA. This material is available free of charge via the Internet at http://pubs.acs.org.

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  • Corresponding Author
    • Danzhou Yang - ‡Department of Pharmacology and Toxicology, College of Pharmacy, †Department of Chemistry, §BIO5 Institute, ∥The Arizona Cancer Center, University of Arizona, 1703 East Mabel Street, Tucson, Arizona 85721, United States
  • Authors
    • Prashansa Agrawal - ‡Department of Pharmacology and Toxicology, College of Pharmacy, †Department of Chemistry, §BIO5 Institute, ∥The Arizona Cancer Center, University of Arizona, 1703 East Mabel Street, Tucson, Arizona 85721, United States
    • Clement Lin - ‡Department of Pharmacology and Toxicology, College of Pharmacy, †Department of Chemistry, §BIO5 Institute, ∥The Arizona Cancer Center, University of Arizona, 1703 East Mabel Street, Tucson, Arizona 85721, United States
    • Raveendra I. Mathad - ‡Department of Pharmacology and Toxicology, College of Pharmacy, †Department of Chemistry, §BIO5 Institute, ∥The Arizona Cancer Center, University of Arizona, 1703 East Mabel Street, Tucson, Arizona 85721, United States
    • Megan Carver - ‡Department of Pharmacology and Toxicology, College of Pharmacy, †Department of Chemistry, §BIO5 Institute, ∥The Arizona Cancer Center, University of Arizona, 1703 East Mabel Street, Tucson, Arizona 85721, United States
  • Notes
    The authors declare no competing financial interest.

Acknowledgment

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This research is supported by the National Institutes of Health (CA122952 and GM083117 to D.Y.).

References

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    BACKGROUND: The bcl-2 gene becomes activated by 14;18 chromosomal translocations in the majority of low-grade non-Hodgkin's lymphomas (NHLs) and is expressed at high levels in the absence of gene rearrangements in a high proportion of B-cell chronic lymphocytic leukemias (B-CLLs). The protein encoded by bcl-2 contributes to neoplastic cell expansion by prolonging cell survival through its ability to block programmed cell death (apoptosis). Because many chemotherapeutic drugs have been shown ultimately to kill tumor cells through mechanisms consistent with programmed cell death, we tested whether the relative levels of bcl-2 oncoprotein influence the sensitivity of lymphoma and leukemia cell lines to killing by conventional cytotoxic drugs commonly used in the treatment of cancer. METHODS: Leukemia cell lines with low levels of bcl-2 expression were stably infected with recombinant bcl-2 retroviruses to achieve elevations in bcl-2 protein levels. Lymphoma cell lines with high levels of bcl-2 expression as the result of 14;18 translocations were either stably transfected with inducible bcl-2 antisense expression plasmids or treated with bcl-2 antisense oligonucleotides to achieve reductions in bcl-2 protein levels. The sensitivity of these genetically modified cells to killing by various antineoplastic drugs was then determined. RESULTS: Gene transfer-mediated elevations in bcl-2 protein levels in lymphocytic leukemia cell lines was correlated with markedly elevated resistance to killing by all cytotoxic drugs tested. Conversely, antisense-mediated reductions in bcl-2 protein levels in t(14;18)-containing NHL cell lines resulted in enhanced sensitivity to all anticancer drugs. CONCLUSIONS: The relative levels of bcl-2 oncoprotein represent one of the key determinants of the sensitivity of lymphocytic cells to killing by essentially all drugs currently available for the treatment of cancer.
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    An inhibitor of Bcl-2 family proteins induces regression of solid tumours
    Oltersdorf, Tilman; Elmore, Steven W.; Shoemaker, Alexander R.; Armstrong, Robert C.; Augeri, David J.; Belli, Barbara A.; Bruncko, Milan; Deckwerth, Thomas L.; Dinges, Jurgen; Hajduk, Philip J.; Joseph, Mary K.; Kitada, Shinichi; Korsmeyer, Stanley J.; Kunzer, Aaron R.; Letai, Anthony; Li, Chi; Mitten, Michael J.; Nettesheim, David G.; Ng, Shi Chung; Nimmer, Paul M.; O'Connor, Jacqueline M.; Oleksijew, Anatol; Petros, Andrew M.; Reed, John C.; Shen, Wang; Tahir, Stephen K.; Thompson, Craig B.; Tomaselli, Kevin J.; Wang, Baole; Wendt, Michael D.; Zhang, Haichao; Fesik, Stephen W.; Rosenberg, Saul H.
    Nature (London, United Kingdom) (2005), 435 (7042), 677-681CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-XL and Bcl-2, are overexpressed in many cancers and contribute to tumor initiation, progression and resistance to therapy. Bcl-XL expression correlates with chemo-resistance of tumor cell lines, and redns. in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anticancer therapeutics has been previously explored, but obtaining potent small-mol. inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using NMR-based screening, parallel synthesis and structure-based design, the authors have discovered ABT-737, a small-mol. inhibitor of the antiapoptotic proteins Bcl-2, Bcl-XL and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumors, and produces cures in a high percentage of the mice.
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    Discovery of Small-Molecule Inhibitors of Bcl-2 through Structure-Based Computer Screening
    Enyedy, Istvan J.; Ling, Yan; Nacro, Kassoum; Tomita, York; Wu, Xihan; Cao, Yeyu; Guo, Ribo; Li, Bihua; Zhu, Xiaofeng; Huang, Ying; Long, Ya-Qiu; Roller, Peter P.; Yang, Dajun; Wang, Shaomeng
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    Bcl-2 belongs to a growing family of proteins which regulates programmed cell death (apoptosis). Overexpression of Bcl-2 has been obsd. in 70% of breast cancer, 30-60% of prostate cancer, 80% of B-cell lymphomas, 90% of colorectal adenocarcinomas, and many other forms of cancer. Thereby, Bcl-2 is an attractive new anti-cancer target. Herein, we describe the discovery of novel classes of small-mol. inhibitors targeted at the BH3 binding pocket in Bcl-2. The three-dimensional (3D) structure of Bcl-2 has been modeled on the basis of a high-resoln. NMR soln. structure of Bcl-XL, which shares a high sequence homol. with Bcl-2. A structure-based computer screening approach has been employed to search the National Cancer Institute 3D database of 206 876 org. compds. to identify potential Bcl-2 small-mol. inhibitors that bind to the BH3 binding site of Bcl-2. These potential Bcl-2 small-mol. inhibitors were first tested in an in vitro binding assay for their potency in inhibition of the binding of a Bak BH3 peptide to Bcl-2. Thirty-five potential inhibitors were tested in this binding assay, and seven of them were found to have a binding affinity (IC50 value) from 1.6 to 14.0 M. The anti-proliferative activity of these seven active compds. has been tested using a human myeloid leukemia cell line, HL-60, which expresses the highest level of Bcl-2 protein among all the cancer cell lines examd. The most potent compd. had an IC50 value of 4 μM in inhibition of cell growth using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Five other compds. had moderate activity in inhibition of cell growth. The most active compd. was further evaluated for its ability to induce apoptosis in cancer cells. It was found that this compd. induces apoptosis in cancer cells with high Bcl-2 expression and its potency correlates with the Bcl-2 expression level in cancer cells. Furthermore, using NMR methods, we conclusively demonstrated that this most active compd. binds to the BH3 binding site in Bcl-XL. Our results showed that small-mol. inhibitors of Bcl-2 modulate the biol. function of Bcl-2, and induce apoptosis in cancer cells with high Bcl-2 expression, while they have little effect on cancer cells with low or undetectable levels of Bcl-2 expression. Therefore, this active compd. can be used as a valuable pharmacol. tool to elucidate the function of Bcl-2 and also serves as a novel lead compd. for further design and optimization. Our results suggest that the structure-based computer screening strategy employed in the study is effective for identifying novel, structurally diverse, nonpeptide small-mol. inhibitors that target the BH3 binding site of Bcl-2.
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    Nature Cell Biology (2001), 3 (2), 183-192CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)
    The Bcl-2-related survival proteins confer cellular resistance to a wide range of agents. Bcl-xL-expressing hepatocyte cell lines are resistant to tumor necrosis factor and anti-cancer drugs, but are more sensitive than isogenic control cells to antimycin A, an inhibitor of mitochondrial electron transfer. Computational mol. docking anal. predicted that antimycin A interacts with the Bcl-2 homol. domain 3 (BH3)-binding hydrophobic groove of Bcl-xL. We demonstrate that antimycin A and a Bak BH3 peptide bind competitively to recombinant Bcl-2. Antimycin A and BH3 peptide both induce mitochondrial swelling and loss of Δψm on addn. to mitochondria expressing Bcl-xL. The 2-methoxy deriv. of antimycin A3 is inactive as an inhibitor of cellular respiration but still retains toxicity for Bcl-xL+ cells and mitochondria. Finally, antimycin A inhibits the pore-forming activity of Bcl-xL in synthetic liposomes, demonstrating that a small non-peptide ligand can directly inhibit the function of Bcl-2-related proteins.
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    Annals of oncology : official journal of the European Society for Medical Oncology / ESMO (2004), 15 (8), 1274-83 ISSN:0923-7534.
    PURPOSE: Expression of the Bcl-2 protein confers resistance to various apoptotic signals. G3139 [oblimersen sodium (Genasense)] is a phosphorothioate antisense oligodeoxynucleotide that targets Bcl-2 mRNA, downregulates Bcl-2 protein translation, and enhances the antitumor effects of subtherapeutic doses of docetaxel (Taxotere). PATIENTS AND METHODS: We performed a phase I trial to determine the maximum tolerated dose (MTD) and safety profile of combined therapy with G3139 and weekly docetaxel in patients with advanced Bcl-2-positive solid tumors. Cohorts of three to six patients were enrolled to escalating doses of G3139 and a fixed dose of weekly docetaxel using either of two schedules. In part I, G3139 was administered by continuous infusion for 21 days (D1-22), and docetaxel (35 mg/m2) was given weekly on days 8, 15 and 22. In part II, G3139 was given by continuous infusion for 5 days before the first weekly dose of docetaxel, and for 48 h before the second and third weekly docetaxel doses. For both schedules, cycles were repeated every 4 weeks. RESULTS: Twenty-two patients were enrolled. Thirteen patients were treated on the part I schedule with doses of G3139 escalated from 1 to 4 mg/kg/day. Nine patients were on the part II schedule of shorter G3139 infusion at G3139 doses of 5-9 mg/kg/day. Hematologic toxicities were mild, except for one case of persistent grade 3 thrombocytopenia in part I. The most common adverse events were cumulative fatigue and transaminase elevation, which prevented further dose escalation beyond 4 mg/kg/day for 21 days with the part I schedule. In part II of the study, using the abbreviated G3139 schedule, even the highest daily doses were tolerated without dose-limiting toxicity or the need for dose modification. Objective tumor response was observed in two patients with breast cancer, including one whose cancer previously progressed on trastuzumab plus paclitaxel. Four patients had stable disease. Pharmacokinetic results for G3139 were similar to those of other trials. CONCLUSIONS: G3139 in combination with standard-dose weekly docetaxel was well tolerated. The shortened and intermittent G3139 infusion had less cumulative toxicities and still allowed similar total G3139 delivery as the longer infusion. Further studies should examine the molecular effect of the regimen, as well as clinical activities in malignancies for which taxanes are indicated.
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Cite this: J. Am. Chem. Soc. 2014, 136, 5, 1750–1753
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  • Abstract

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    Figure 1

    Figure 1. (A) The promoter sequence of the BCL-2 gene and its modifications. The top sequence is the 39-mer wild-type G-rich sequence (Pu39). The six G-runs with three or more guanines are underlined and numbered. Pu30 is the wild-type 30-mer G-rich sequence containing the I–V G-runs; the numbering used in this study is shown for Pu30. The guanine residues that are involved in the tetrad formation of the major BCL-2 G-quadruplex 1245G4 are shown in red. The mutations are shown in cyan. (B) DMS footprinting of the wild-type Pu39 with densitometric scans (left) and Pu30 (right). (C) Imino regions of 1D 1H NMR spectra of BCL-2 promoter sequences at 25 °C in 45 mM K+, pH 7.0. (D) CD spectra of Pu30 sequences in 95 mM K+.

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    Figure 2

    Figure 2. (A) A G-tetrad with detectable H1–H1 and H1–H8 NOE connectivity. (B) Imino H1 and aromatic H8 proton assignments of Pu30_3T4AA by 1D 15N-filtered experiments using site-specific labeled oligonucleotides at 25 °C. (C) Schematic drawing of the folding topology of the major G-quadruplex 1245G4 formed in the BCL-2 promoter sequence Pu30 (G = red, A = green, C = yellow). (D) H1–H1 region and (E) H1–H8 region of the 2D-NOESY spectrum of Pu30_3T4AA in H2O at 5 °C with a mixing time of 200 ms. The proton assignments are shown on the sides. Intratetrad NOEs are in red, intertetrad NOEs in blue, sequential intertetrad NOEs in black, and NOEs with flanking bases in green. Conditions: 25 mM K-phosphate, 70 mM KCl, pH 7.0.

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    Figure 3

    Figure 3. G-quadruplex-forming promoter sequences.

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      Marshall J; Chen H; Yang D; Figueira M; Bouker K B; Ling Y; Lippman M; Frankel S R; Hayes D F
      Annals of oncology : official journal of the European Society for Medical Oncology / ESMO (2004), 15 (8), 1274-83 ISSN:0923-7534.
      PURPOSE: Expression of the Bcl-2 protein confers resistance to various apoptotic signals. G3139 [oblimersen sodium (Genasense)] is a phosphorothioate antisense oligodeoxynucleotide that targets Bcl-2 mRNA, downregulates Bcl-2 protein translation, and enhances the antitumor effects of subtherapeutic doses of docetaxel (Taxotere). PATIENTS AND METHODS: We performed a phase I trial to determine the maximum tolerated dose (MTD) and safety profile of combined therapy with G3139 and weekly docetaxel in patients with advanced Bcl-2-positive solid tumors. Cohorts of three to six patients were enrolled to escalating doses of G3139 and a fixed dose of weekly docetaxel using either of two schedules. In part I, G3139 was administered by continuous infusion for 21 days (D1-22), and docetaxel (35 mg/m2) was given weekly on days 8, 15 and 22. In part II, G3139 was given by continuous infusion for 5 days before the first weekly dose of docetaxel, and for 48 h before the second and third weekly docetaxel doses. For both schedules, cycles were repeated every 4 weeks. RESULTS: Twenty-two patients were enrolled. Thirteen patients were treated on the part I schedule with doses of G3139 escalated from 1 to 4 mg/kg/day. Nine patients were on the part II schedule of shorter G3139 infusion at G3139 doses of 5-9 mg/kg/day. Hematologic toxicities were mild, except for one case of persistent grade 3 thrombocytopenia in part I. The most common adverse events were cumulative fatigue and transaminase elevation, which prevented further dose escalation beyond 4 mg/kg/day for 21 days with the part I schedule. In part II of the study, using the abbreviated G3139 schedule, even the highest daily doses were tolerated without dose-limiting toxicity or the need for dose modification. Objective tumor response was observed in two patients with breast cancer, including one whose cancer previously progressed on trastuzumab plus paclitaxel. Four patients had stable disease. Pharmacokinetic results for G3139 were similar to those of other trials. CONCLUSIONS: G3139 in combination with standard-dose weekly docetaxel was well tolerated. The shortened and intermittent G3139 infusion had less cumulative toxicities and still allowed similar total G3139 delivery as the longer infusion. Further studies should examine the molecular effect of the regimen, as well as clinical activities in malignancies for which taxanes are indicated.
    16. 16
      Klasa, R. J.; Gillum, A. M.; Klem, R. E.; Frankel, S. R. Antisense Nucleic Acid Drug Dev. 2002, 12, 193– 213
      There is no corresponding record for this reference.
    17. 17
      Rantanen, S.; Monni, O.; Joensuu, H.; Franssila, K.; Knuutila, S. Leuk. Lymphoma 2001, 42, 1089– 1098
      17
      Causes and consequences of BCL2 overexpression in diffuse large B-cell lymphoma
      Rantanen, Sari; Monni, Outi; Joensuu, Heikki; Franssila, Kaarle; Knuutila, Sakari
      Leukemia & Lymphoma (2001), 42 (5), 1089-1098CODEN: LELYEA; ISSN:1042-8194. (Harwood Academic Publishers)
      We investigated the frequency of bcl-2 protein overexpression in 80 diffuse large B-cell lymphoma (DLBCL) patients using both Western blotting and immunohistochem. (IHC). Fifty-nine percent of the DLBCLs overexpressed bcl-2 protein by Western blot and 52% by IHC. The 2 methods usually gave concordant results, but 14 (21%) out of the 67 cases that were analyzed by both methods were pos. by Western blot and neg. by IHC, and 8 (12%) cases vice versa. Bcl-2 overexpression by IHC was assocd. with poor response to chemotherapy and poor survival, whereas these assocns. were not found when bcl-2 overexpression was detd. by Western blotting. The mol. mechanisms leading to bcl-2 overexpression were evaluated by PCR, karyotype anal., and comparative genomic hybridization (CGH). When studied by PCR and/or karyotype anal., 12 (15%) of the 80 cases had translocation (14;18)(q32;q21). All 12 lymphomas with (14;18)(q32;q21) translocation had bcl-2 overexpression by Western blot as compared with 35 (51%) of the 68 lymphomas without translocation. Ten (29%) out of 34 cases that were analyzed by CGH showed amplification of chromosome 18 in which the BCL2 gene is located, and all cases showed bcl-2 overexpression by both Western blot and IHC. The results suggest that gene amplification and translocation are at least equally common mechanisms causing bcl-2 protein overexpression in DLBCL. Bcl-2 protein overexpression as detd. by IHC is assocd. with poor response to chemotherapy and poor survival.
    18. 18
      Seto, M.; Jaeger, U.; Hockett, R. D.; Graninger, W.; Bennett, S.; Goldman, P.; Korsmeyer, S. J. EMBO J. 1988, 7, 123– 131
      There is no corresponding record for this reference.
    19. 19
      Young, R. L.; Korsmeyer, S. J. Mol. Cell. Biol. 1993, 13, 3686– 3697
      19
      A negative regulatory element in the bcl-2 5'-untranslated region inhibits expression from an upstream promoter
      Young, Robert L.; Korsmeyer, Stanley J.
      Molecular and Cellular Biology (1993), 13 (6), 3686-97CODEN: MCEBD4; ISSN:0270-7306.
      Bcl-2 mRNA is present at high levels in pre-B-cell lines but is down-regulated in most mature B-cell lines. To investigate the mechanisms responsible for its developmental control, the regulation of bcl-2 expression was studied in human B-lineage cell lines. Using nuclear run-on assays, it was found that bcl-2 transcription decreases in parallel with levels of steady-state mRNA during B-cell development. To define cis-acting elements that regulate bcl-2 transcription, the expression of transiently transfected promoter-reporter constructs was analyzed. A novel neg. regulatory element (NRE) was identified in the bcl-2 5'-untranslated region that decreased expression from the bcl-2 P1 promoter or heterlogous promoters in a position-dependent fashion. The NRE functions in either orientation but contains distinct orientation-dependent subfragments. Addnl. analyses demonstrated that multiple, functionally redundant sequence elements mediate NRE activity. Though the bcl-2 NRE is active in pre-B- and mature B-cell lines, chromatin structure of the endogenous NRE differs in these cells, suggesting that its activity or effect may vary during B-cell development. The results indicate that neg. control of transcription initiated at the P1 promoter is an important determinant of the differential expression of bcl-2.
    20. 20
      Heckman, C.; Mochon, E.; Arcinas, M.; Boxer, L. M. J. Biol. Chem. 1997, 272, 19609– 19614
      There is no corresponding record for this reference.
    21. 21
      Gomez-Manzano, C.; Mitlianga, P.; Fueyo, J.; Lee, H. Y.; Hu, M. Cancer Res. 2001, 61, 6693– 6697
      There is no corresponding record for this reference.
    22. 22
      Liu, Y. Z.; Boxer, L. M.; Latchman, D. S. Nucleic Acids Res. 1999, 27, 2086– 2090
      There is no corresponding record for this reference.
    23. 23
      Dai, J.; Dexheimer, T. S.; Chen, D.; Carver, M.; Ambrus, A.; Jones, R. A.; Yang, D. Z. J. Am. Chem. Soc. 2006, 128, 1096– 1098
      There is no corresponding record for this reference.
    24. 24
      Dexheimer, T. S.; Sun, D.; Hurley, L. H. J. Am. Chem. Soc. 2006, 128, 5404– 5415
      There is no corresponding record for this reference.
    25. 25
      Onyshchenko, M. I.; Gaynutdinov, T. I.; Englund, E. A.; Appella, D. H.; Neumann, R. D.; Panyutin, I. G. Nucleic Acids Res. 2009, 37, 7570– 7580
      There is no corresponding record for this reference.
    26. 26
      Dai, J.; Chen, D.; Jones, R. A.; Hurley, L. H.; Yang, D. Z. Nucleic Acids Res. 2006, 34, 5133– 5144
      26
      NMR solution structure of the major G-quadruplex structure formed in the human BCL2 promoter region
      Dai, Jixun; Chen, Ding; Jones, Roger A.; Hurley, Laurence H.; Yang, Danzhou
      Nucleic Acids Research (2006), 34 (18), 5133-5144CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      BCL2 protein functions as an inhibitor of cell apoptosis and has been found to be aberrantly expressed in a wide range of human diseases. A highly GC-rich region upstream of the P1 promoter plays an important role in the transcriptional regulation of BCL2. Here we report the NMR soln. structure of the major intramol. G-quadruplex formed on the G-rich strand of this region in K+ soln. This well-defined mixed parallel/antiparallel-stranded G-quadruplex structure contains three G-tetrads of mixed G-arrangements, which are connected with two lateral loops and one side loop, and four grooves of different widths. The three loops interact with the core G-tetrads in a specific way that defines and stabilizes the overall G-quadruplex structure. The loop conformations are in accord with the exptl. mutation and footprinting data. The first 3-nt loop adopts a lateral loop conformation and appears to det. the overall folding of the BCL2 G-quadruplex. The third 1-nt double-chain-reversal loop defines another example of a stable parallel-stranded structural motif using the G3NG3 sequence. Significantly, the distinct major BCL2 promoter G-quadruplex structure suggests that it can be specifically involved in gene modulation and can be an attractive target for pathway-specific drug design.
    27. 27
      Siddiqui-Jain, A.; Grand, C. L.; Bearss, D. J.; Hurley, L. H. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11593– 11598
      27
      Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription
      Siddiqui-Jain, Adam; Grand, Cory L.; Bearss, David J.; Hurley, Laurence H.
      Proceedings of the National Academy of Sciences of the United States of America (2002), 99 (18), 11593-11598CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The nuclease hypersensitivity element III1 upstream of the P1 promoter of c-MYC controls 85-90% of the transcriptional activation of this gene. We have demonstrated that the purine-rich strand of the DNA in this region can form two different intramol. G-quadruplex structures, only one of which seems to be biol. relevant. This biol. relevant structure is the kinetically favored chairform G-quadruplex, which is destabilized when mutated with a single G → A transition, resulting in a 3-fold increase in basal transcriptional activity of the c-MYC promoter. The cationic porphyrin TMPyP4, which has been shown to stabilize this G-quadruplex structure, is able to suppress further c-MYC transcriptional activation. These results provide compelling evidence that a specific G-quadruplex structure formed in the c-MYC promoter region functions as a transcriptional repressor element. Furthermore, we establish the principle that c-MYC transcription can be controlled by ligand-mediated G-quadruplex stabilization.
    28. 28
      Agrawal, P.; Hatzakis, E.; Guo, K.; Carver, M.; Yang, D. Nucleic Acids Res. 2013, 41, 10584– 10592
      There is no corresponding record for this reference.
    29. 29
      Hatzakis, E.; Okamoto, K.; Yang, D. Z. Biochemistry 2010, 49, 9152– 9160
      There is no corresponding record for this reference.
    30. 30
      Balasubramanian, S.; Hurley, L. H.; Neidle, S. Nat. Rev. Drug Discovery 2011, 10, 261– 275
      30
      Targeting G-quadruplexes in gene promoters: a novel anticancer strategy?
      Balasubramanian, Shankar; Hurley, Laurence H.; Neidle, Stephen
      Nature Reviews Drug Discovery (2011), 10 (4), 261-275CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
      A review. G-quadruplexes are four-stranded DNA structures that are over-represented in gene promoter regions and are viewed as emerging therapeutic targets in oncol., as transcriptional repression of oncogenes through stabilization of these structures could be a novel anticancer strategy. Many gene promoter G-quadruplexes have physicochem. properties and structural characteristics that might make them druggable, and their structural diversity suggests that a high degree of selectivity might be possible. Here, we describe the evidence for G-quadruplexes in gene promoters and discuss their potential as therapeutic targets, as well as progress in the development of strategies to harness this potential through intervention with small-mol. ligands.
    31. 31
      Ambrus, A.; Chen, D.; Dai, J.; Jones, R. A.; Yang, D. Z. Biochemistry 2005, 44, 2048– 2058
      There is no corresponding record for this reference.
    32. 32
      Seenisamy, J.; Rezler, E. M.; Powell, T. J.; Tye, D.; Gokhale, V.; Joshi, C. S.; Siddiqui-Jain, A.; Hurley, L. H. J. Am. Chem. Soc. 2004, 126, 8702– 8709
      There is no corresponding record for this reference.
    33. 33
      Phan, A. T.; Modi, Y. S.; Patel, D. J. J. Am. Chem. Soc. 2004, 126, 8710– 8716
      33
      Propeller-Type Parallel-Stranded G-Quadruplexes in the Human c-myc Promoter
      Phan, Anh Tuan; Modi, Yasha S.; Patel, Dinshaw J.
      Journal of the American Chemical Society (2004), 126 (28), 8710-8716CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The nuclease-hypersensitivity element III1 in the c-myc promoter is a good anticancer target since it largely controls transcriptional activation of the important c-myc oncogene. Recently, the guanine-rich strand of this element has been shown to form an equil. between G-quadruplex structures built from two different sets of G-stretches; two models of intramol. fold-back antiparallel-stranded G-quadruplexes, called "basket" and "chair" forms, were proposed. Here, the authors show by NMR that two sequences contg. these two sets of G-stretches form intramol. propeller-type parallel-stranded G-quadruplexes in K+-contg. soln. The two structures involve a core of three stacked G-tetrads formed by four parallel G-stretches with all anti guanines and three double-chain-reversal loops bridging three G-tetrad layers. The central loop contains two or six residues, while the two other loops contain only one residue.
    34. 34
      Hsu, S. T.; Varnai, P.; Bugaut, A.; Reszka, A. P.; Neidle, S.; Balasubramanian, S. J. Am. Chem. Soc. 2009, 131, 13399– 13409
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    35. 35
      De Armond, R.; Wood, S.; Sun, D. Y.; Hurley, L. H.; Ebbinghaus, S. W. Biochemistry 2005, 44, 16341– 16350
      There is no corresponding record for this reference.
    36. 36
      Guo, K.; Pourpak, A.; Beetz-Rogers, K.; Gokhale, V.; Sun, D.; Hurley, L. H. J. Am. Chem. Soc. 2007, 129, 10220– 10228
      There is no corresponding record for this reference.
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      Palumbo, S. L.; Ebbinghaus, S. W.; Hurley, L. H. J. Am. Chem. Soc. 2009, 131, 10878– 10891
      There is no corresponding record for this reference.
    38. 38
      Lim, K. W.; Lacroix, L.; Yue, D. J. E.; Lim, J. K. C.; Lim, J. M. W.; Phan, A. T. J. Am. Chem. Soc. 2010, 132, 12331– 12342
      There is no corresponding record for this reference.
    39. 39
      Chen, Y.; Yang, D. Z. Curr. Protoc. Nucleic Acid Chem. 2012, 50, 17.15.11– 17.15.17
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      Rachwal, P. A.; Brown, T.; Fox, K. R. FEBS Lett. 2007, 581, 1657– 1660
      There is no corresponding record for this reference.
    41. 41
      Todd, A. K.; Johnston, M.; Neidle, S. Nucleic Acids Res. 2005, 33, 2901– 2907
      There is no corresponding record for this reference.
    42. 42
      Bugaut, A.; Balasubramanian, S. Biochemistry 2008, 47, 689– 697
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    43. 43
      Mathad, R. I.; Hatzakis, E.; Dai, J.; Yang, D. Z. Nucleic Acids Res. 2011, 39, 9023– 9033
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    44. 44
      Huppert, J. L.; Balasubramanian, S. Nucleic Acids Res. 2005, 33, 2908– 2916
      44
      Prevalence of quadruplexes in the human genome
      Huppert, Julian L.; Balasubramanian, Shankar
      Nucleic Acids Research (2005), 33 (9), 2908-2916CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      Guanine-rich DNA sequences of a particular form have the ability to fold into four-stranded structures called G-quadruplexes. In this paper, the authors present a working rule to predict which primary sequences can form this structure, and describe a search algorithm to identify such sequences in genomic DNA. The authors count the no. of quadruplexes found in the human genome and compare that with the figure predicted by modeling DNA as a Bernoulli stream or as a Markov chain, using windows of various sizes. The authors demonstrate that the distribution of loop lengths is significantly different from what would be expected in a random case, providing an indication of the no. of potentially relevant quadruplex-forming sequences. In particular, the authors show that there is a significant repression of quadruplexes in the coding strand of exonic regions, which suggests that quadruplex-forming patterns are disfavored in sequences that will form RNA.
    45. 45
      Guédin, A.; Gros, J.; Alberti, P.; Mergny, J.-L. Nucleic Acids Res. 2010, 38, 7858– 7868
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    Materials and methods, 1D 1H NMR spectra of Pu39 and Pu30 sequences, and H8/H6–H1′ region of the nonexchangeable 2D-NOESY, CD melting and NMR VT studies, of Pu30_3T4AA. This material is available free of charge via the Internet at http://pubs.acs.org.


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