Indenoisoquinoline Topoisomerase Inhibitors Strongly Bind and Stabilize the MYC Promoter G-Quadruplex and Downregulate MYC

Abstract

MYC is one of the most important oncogenes and is overexpressed in the majority of cancers. G-Quadruplexes are noncanonical four-stranded DNA secondary structures that have emerged as attractive cancer-specific molecular targets for drug development. The G-quadruplex formed in the proximal promoter region of the MYC oncogene (MycG4) has been shown to be a transcriptional silencer that is amenable to small-molecule targeting for MYC suppression. Indenoisoquinolines are human topoisomerase I inhibitors in clinical testing with improved physicochemical and biological properties as compared to the clinically used camptothecin anticancer drugs topotecan and irinotecan. However, some indenoisoquinolines with potent anticancer activity do not exhibit strong topoisomerase I inhibition, suggesting a separate mechanism of action. Here, we report that anticancer indenoisoquinolines strongly bind and stabilize MycG4 and lower MYC expression levels in cancer cells, using various biochemical, biophysical, computer modeling, and cell-based methods. Significantly, a large number of active indenoisoquinolines cause strong MYC downregulation in cancer cells. Structure-activity relationships of MycG4 recognition by indenoisoquinolines are investigated. In addition, the analysis of indenoisoquinoline analogues for their MYC-inhibitory activity, topoisomerase I-inhibitory activity, and anticancer activity reveals a synergistic effect of MYC inhibition and topoisomerase I inhibition on anticancer activity. Therefore, this study uncovers a novel mechanism of action of indenoisoquinolines as a new family of drugs targeting the MYC promoter G-quadruplex for MYC suppression. Furthermore, the study suggests that dual targeting of MYC and topoisomerase I may serve as a novel strategy for anticancer drug development.

Figure 1.

Chemical structures of indenoisoquinoline topoisomerase I inhibitors in Phase I clinical trials and quindoline, as well as the MYC promoter and MYC promoter G-quadruplex. (A) Indenoisoquinoline topoisomerase I inhibitors currently in clinical trials. (B) Left: The structure of the human MYC gene promoter. The G4-forming region NHE III₁ sequence is shown, with the guanine runs underlined. The guanine runs involved in the formation of the major MycG4 are highlighted in red. Right: The folding topology of MycG4 adopted by the MycPu22 sequence is a parallel-stranded 3-tetrad G-quadruplex, with the two stabilizing potassium cations shown. Red ball = guanine, green ball = adenine, blue ball = thymine, large blue ball = K⁺. (C) Left: a MycG4 stabilizer quindoline and a topoisomerase I inhibitor indenoisoquinoline. Right: overlay of the three-dimensional structures of quindoline and an indenoisoquinoline in their energy-minimized states.

Figure 2.

Indenoisoquinolines can induce and stabilize MycG4. (A) Left: schematic of the FRET-quenching assay used for compound screening. The FRET-quenching (shown as fluorophore in black color) caused by MycG4 folding can be induced by K⁺ or MycG4-inducing compounds. Right: relative fluorescence intensities of the labeled MycG4 in the presence of DMSO, 100 mM K⁺, and indenoisoquinoline analogs as shown by FRET-quenching assay. Data shown are the average values of the two individual experiments. DMSO (negative control), 100 mM K⁺ (positive control), and six indenoisoquinolines used for further studies are highlighted and labeled. Conditions: 1 μM labeled DNA, 10 μM compound, 25 °C, 50 mM Tris·acetate, pH 7. (B) Thermal stabilization values (ΔT_m) of MycG4 by indenoisoquinoline analogs as shown by FRET-melting assay. Data shown are the average values of the two individual experiments. The six representative indenoisoquinolines used for further studies are highlighted and labeled. Conditions: 150 nM labeled DNA, 1.5 μM compound, 25 °C, pH 7, 10 mM K⁺. (C) Correlation of FRET-quenching and FRET-melting data. The Pearson correlation coefficient (r) is shown.

Figure 3.

MYC inhibitory activities of indenoisoquinoline analogs. (A) MYC protein expression levels in the absence and presence of various concentrations of indenoisoquinolines (24 hr treatment) were obtained by western blotting experiments in MCF-7 breast cancer cells. GAPDH was used as an internal control. (B) Plot of the topoisomerase I inhibition levels against the MGM values of 31 indenoisoquinolines that were used to determine topoisomerase, MYC, and MGM activities. The yellow shaded area indicated the region of more active indenoisoquinolines. Based on the MYC downregulation shown in the western blotting results (Figures 3A and S3), MYC inhibition levels were classified into four levels: strong inhibition, MYC expression inhibited at 0.5 to 1.0 μM, red dots; medium inhibition, MYC expression inhibited at 2.0 μM or no clear dose-dependent MYC inhibition, pink dots; weak inhibition, MYC expression inhibited at 4.0 μM, blue dots; no inhibition, no MYC expression inhibition up to 4.0 μM, black dots. The relative topoisomerase I (Top1) inhibition levels of the compounds were previously determined and classified into six levels (0 – 5).^{, –, , –} The MGM values are the average of GI₅₀ values across the entire panel of NCI-60 cancer cell lines; the GI₅₀ values are the concentrations corresponding to 50% growth inhibition which were determined in the NCI-60 cancer cell lines drug screen (Table S3, Figures S8 and S9). (C) MYC transcription levels in the absence and presence of indenoisoquinolines (6 hr treatment) were obtained by qRT-PCR experiments in MCF-7 cancer cells. DMSO was used as the negative control (no inhibition, 100%). The relative MYC mRNA levels were normalized with GAPDH. The experiments were run in triplicate. P values: ***P < 0.0004, ****P < 0.0001.

Figure 4.

SAR of selected indenoisoquinolines. N.D., not determined.

Figure 5.

1D ¹H NMR titrations of MycPu22 DNA with indenoisoquinolines and 7-azaindenoisoquinolines. Imino proton regions of the titration spectra of MycG4 with compound 5 (A), 6 (B), 13 (C), 9 (D), 12 (E), and 17 (F) are shown. In Figure 5A, the imino proton signals from the 5′ G-tetrad (Figure 1B) are labeled in red, the middle G-tetrad in black, and the 3′ G-tetrad in green. Conditions: 150 μM DNA, 25 °C, pH 7, 100 mM K⁺.

Figure 6.

A model of the 2:1 complex of 7-azaindenoisoquinoline 5:MycG4 suggested by Glide docking in different views. 7-azaindenoisoquinoline 5 is shown in green. Intermolecular salt bridges are shown as black dashed lines.

Figure 7.

Binding selectivities of MycG4-interactive indenoisoquinolines. Competition fluorescence displacement experiments with increasing concentrations of unlabeled G4s and dsDNA added to 3′-TAMRA-labeled MycPu22 (20 nM) mixed with 1 equivalent of compound 13 (A), 5 (B), 6 (C), 9 (D), and 12 (E). The normalized TAMRA fluorescence intensities at 580 nm were plotted as a function of molar ratio of added G4 DNA (in 3 G-tetrads) or calf thymus dsDNA (in 11 bp) to labeled MycPu22 DNA. The fluorescence intensity of free 3′-TAMRA labeled MycPu22 was defined as 100%, and 1:1 mixture of 3′-TAMRA labeled MycPu22 and indenoisoquinoline was defined as 0%. Conditions: 20 °C, pH 7, 100 mM K⁺.

Figure 8.

(A) A schematic model showing the potential mechanisms of MYC suppression by indenoisoquinolines by (a) stabilization of MycG4 in the MYC promoter to inhibit transcription, and (b) inhibition of topoisomerase I to maintain negative supercoiling for G4 formation. (B) A heat map showing the synergistic effect of MYC inhibition and topoisomerase I inhibition on the anticancer activities of 29 indenoisoquinolines. The 29 indenoisoquinolines are grouped by their MYC inhibition levels and topoisomerase I inhibition levels. The anticancer activity for each group is determined by the mean(log₁₀MGM) value of the grouped compounds (Table S4), which is displayed as color gradient in the heat map. The MGM values are the approximate average of GI₅₀ values across the entire panel of NCI-60 cancer cell lines for each compound (Table S3). The synergistic effect of MYC inhibition and topoisomerase I inhibition is reflected by the increased anticancer activities (redder color) towards the bottom left comer with strong MYC and topoisomerase I inhibitory activities.