A targeting modality for destruction of RNA polymerase I that possesses anticancer activity

Abstract

We define the activity and mechanisms of action of a small molecule lead compound for cancer targeting. We show that the compound, BMH-21, has wide and potent antitumorigenic activity across NCI60 cancer cell lines and represses tumor growth in vivo. BMH-21 binds GC-rich sequences, which are present at a high frequency in ribosomal DNA genes, and potently and rapidly represses RNA polymerase I (Pol I) transcription. Strikingly, we find that BMH-21 causes proteasome-dependent destruction of RPA194, the large catalytic subunit protein of Pol I holocomplex, and this correlates with cancer cell killing. Our results show that Pol I activity is under proteasome-mediated control, which reveals an unexpected therapeutic opportunity.

Copyright © 2014 Elsevier Inc. All rights reserved.

Figures

Figure 1. BMH-21 Inhibits Replication and Viability of Cancer cells In Vitro and Tumor Growth In Vivo

(A) NCI Developmental Therapeutics Program NCI60 screen where 100% represents growth in control cultures and 0% represents initial plating density. (B) Box plot of IC50 values of BMH-21 in NCI60 cell lines and normal human primary cells (as adopted from Peltonen et al., 2010). (C) Box plot of GI50 values of TP53 wild-type and mutant cell lines in the NCI60 panel. (D–F) Flow cytometry analysis of A375 melanoma cells treated with BMH-21 (0.5 μM) for 24 hr. Replicating cells were labeled with 5-BrdU (S). (D) Representative experiment is shown. (E) Cell cycle distribution of data in D. The mean (n = 2) and SD are shown. ns, non-significant, *p 1 is shown. (G, H) Tumor xenografts. (G) A375 melanoma xenograft assay. BMH-21 was injected i.p. on a cycle 6 days on, one day off at the indicated doses. Inset: After the treatments, tumors were stained for Ki67. Scale bar 50 μm. (H) HCT116 colorectal carcinoma xenograft assay. Mice were treated daily with 50 mg/kg BMH-21. Treatments were initiated on established tumors 8 – 13 days post-implantation. p values were determined using Two-way ANOVA. Error bars ±SEM. ****p < 0.0001. See also Figure S1.

Figure 2. Binding and Modeling of BMH-21 with GC-rich DNA

(A) Structures of BMH-21 and BMH-21a1. (B) FID assay of BMH-21 and BMH-21a1 using random deoxyoligonucleotide hairpins. Data is displayed in ranking order and median values of triplicates are shown. (C) Molecular modeling of protonated BMH-21 (left) and BMH-21a1 (right) with DNA hexamer (PDB 1Z3F) using Autodock 4.2. C, cytosine; G, guanine. (D) p53 western blotting analysis. U2OS cells were treated with BMH-21 (1 μM) or BMH-21a1 (1 μM) for 8 hr. See also Figure S2.

Figure 3. BMH-21 Binds rDNA Sequences in GC-content Dependent Mannerand Inhibits rRNA Synthesis In Vitro

(A) rDNA GC-content (top). Red line displays average human genomic DNA GC-content. Locations of randomly picked deoxyoligonucleotides used in the FID assay and rDNA domains are shown (bottom, blue dots). (B) FID assay of BMH-21 using rDNA deoxyoligonucleotides. EtBr represents 100%. The mean (n = 3) and SD are shown. The location of sequences and rDNA domains are shown. Numbering according to Genbank #U13369. (C) Correlation of rDNA sequence GC-content to fluorescence displacement by BMH-21. Pearson correlation coefficient r = −0.963. (D) In vitro Pol I transcription assay. HeLa nuclear extract was mixed with Pol I promoter (pHrP2) plasmid, BMH-21 or ActD, and in vitro transcription product was detected by RT-PCR. The mean (n = 2) and SEM are shown. Controls include full transcription reaction (ctrl), omission of nuclear extract (no extract ctrl), and full reaction with omission of PCR primers (neg. PCR ctrl).

Figure 4. BMH-21 Represses rRNA Synthesis and Causes Segregation of Nucleolar Proteins

(A) A375 cells were treated with BMH-21 (1 μM) for 3 hr and de novo rRNA synthesis was measured using EU labeling and quantified. The mean (n = 2) and SD are shown. (B) A375 cells were treated with BMH-21 (1 μM) for 3 hr and rDNA transcription was analyzed by qPCR using three primer sets for short-lived 5′ETS rRNA. The mean (n = 3) and SD are shown. p as indicated. (C–D) Metabolic labeling using 3H-uridine. A375 cells were treated with BMH-21 at concentrations shown for 4 hr and labeled for the last 2 hr. Total RNA was resolved by electrophoresis and de novo synthesized rRNA was detected using autoradiography. Total 18S rRNA indicates loading.(D) Quantification of 47S rRNA precursor. The mean (n = 2) and ±SD are shown. (E) 3H-uridine pulse chase assay. A375 cells were labeled for 30 min with 3H-uridine, washed and incubated for the indicated times with BMH-21 (1 μM). RNA was isolated and analyzed by agarose gel electrophoresis. (F–G) A375 cells were treated with BMH-21 (1 μM) for 3 hr and stained for UBF, FBL, NPM and NCL. Arrowheads indicate nucleolar caps and asterisks nucleoplasmic translocation. Scale bars 10 μm. (G) Western blotting analyses of respective proteins. See also Figure S3.

Figure 5. BMH-21 Causes Destruction of RPA194

(A) A375 cells were treated with BMH-21 (1 μM) or ActD (50 ng/ml) for 3 hr and stained for RPA194 and NCL. Merged image with DNA staining is shown below. Scale bar 10 μm. (B–C) Western blotting of lysates from BMH-21-treated A375 cells (1 μM, 3 hr) for (B) RPA194, RPA135, TIF-IA and SL1 protein TAFI110 and (C) Pol I complex proteins RPA43 and PAF53. (D) ChIP analyses of RPA194, RPA135 and UBF binding to rDNA of A375 cells mock-treated (DMSO, blue bars) or treated with BMH-21 (1 μM) (red bars) for 3 hr. The mean (n = 3) and SEM are shown. Primer locations based on #U13369 are shown below. (E, F) Kinetics of the nucleolar responses. A375 cells were treated with BMH-21 (1 μM) for times shown. (E) Co-treatment of cells with FUrd, and detection of FUrd incorporation. α-amanitin (2 μM) was used to control Pol II-mediated transcription. (F) Staining for RPA194, green; NCL, red; DNA, blue. Arrowheads, cap structures. Scale bars 10 μm. (G, H) RPA194 half-life analyses. (G) A375 cells were treated with cycloheximide in the presence or absence of BMH-21 (1 μM) for the indicated times. RPA194 was detected using western blotting. NCL, loading control. Representative experiment is shown. (H) Quantification of RPA194 half-life. CHX, cycloheximide. n = 7 independent experiments. Error bars, SD. See also Figure S4.

Figure 6. Loss of RPA194 Correlates with Decreased Cancer Cell Viability

(A) U2OS and RPMI-7951 cells were treated with BMH-21 (1 μM) and ActD (50 ng/ml) for indicated times. Representative western blots for RPA194 and NCL. (B) WS-1 normal human diploid fibroblasts were incubated with BMH-21 (1 μM) or ActD (50 ng/ml) for the indicated times. (C, D) WS-1 normal fibroblasts (C) and RPMI-7951 melanoma cells (D) were treated with BMH-21 (1 μM) for 8 and 6 hr, respectively, and stained for the indicated proteins. Arrowheads, cap structures. Scale bars 10 μm. (E) Correlation analysis of viability and nascent rRNA synthesis. Cells were treated with BMH-21 (1 μM) for 3 hr and incubated for the last 1 hr with EU. Fold change in EU incorporation is plotted. Data represents mean of n = 2. Cell viability was assessed using WST-1 assay following 24 hr treatment with BMH-21. The mean of n = 3 are shown. Pearson correlation coefficient r = 0.471. (F) Cancer cell line responses to BMH-21. Cells were treated with BMH-21 (1 μM) for 3 hr and RPA194 was quantified from western blots. Cell viability was assessed using WST-1 assay after 24 hr treatment. Data represents mean of n = 3 biological repeats for each approach. Pearson correlation coefficient r = 0.882. (G–H) Cell viability and growth in RPA194 depleted cells. U2OS cells were transfected with control (si ctrl) or two different RPA194 targeting RNAi duplexes (si403, si405). Cells were incubated for 48 hr and cell numbers were counted (G) or for 72 hr and cell viability was determined by WST-1 assay (H). n = 2–3, error bars represent SD. **p

Figure 7. Inhibition of RPA194 Degradation does not Rescue Transcription

(A, B) A375 cells were pretreated with MG132 (10 μM) or lactacystin (2 μM) for 30 min and BMH-21 (1 μM) was added for 3 hr. (A) Confocal microscopy, (B) western blotting. (C, D) Inhibition of RPA194 degradation does not rescue transcription. A375 cells were treated with BMH-21 for the indicated times (C) or for 3 hr (D). Cells were co-treated with MG132 as shown. Pol I transcription was measured with 3H-uridine metabolic labeling (C) or FUrd (D) for 1 hr. Scale bars 10 μm. See also Figure S6.

Figure 8. BMH-21 Causes Proteasome-mediated Degradation of RPA194

(A) RPA194 was immunoprecipitated from A375 cell lysates following the indicated treatments, and conjugated ubiquitin (right panel) and input RPA194 (left panel) were detected. (B, C) In vitro degradation assay. In vitro translated RPA194-HA was incubated with BMH-21-treated cell extract and ubiquitination reaction mixture with or without MG132 (MG), and RPA194-HA was detected. Asterisks indicated high-molecular weight bands. (C) Quantification of RPA194-HA. The mean (n = 4) and SD are shown. (D, E) U2OS cells were transfected with USP36-Myc or HAUSP-FLAG plasmids, treated with BMH-21 for 3 hr and co-stained for RPA194 and respective tag antibodies. Expression of RPA194 (red) and USP36-Myc (green) are shown. Arrowhead, nucleolar cap.(E) The percentage of RPA194 positive cells was counted. The average (n = 2 experiments, each with > 100 cells) and SD are shown. ns non-significant, **p < 0.01, ***p < 0.001. Scale bar 10 μm. (F) A375 cells were treated with BMH-21 (1 μM) for 3 hr followed by western blotting for Pol II subunit Rpb1, RPA194 and NCL. (G, H) Effect of UVC-radiation on RPA194. Western blotting of HeLa cells (G) and immunofluorescence analysis (H) of A375 cells 3 hr after UVC treatment. Scale bar 10 μm. See also Figure S7.