Inhibition of BET bromodomain proteins as a therapeutic approach in prostate cancer

Abstract

BET (bromodomain and extra-terminal) proteins regulate gene expression through their ability to bind to acetylated chromatin and subsequently activate RNA PolII-driven transcriptional elongation. Small molecule BET inhibitors prevent binding of BET proteins to acetylated histones and inhibit transcriptional activation of BET target genes. BET inhibitors attenuate cell growth and survival in several hematologic cancer models, partially through the down-regulation of the critical oncogene, MYC. We hypothesized that BET inhibitors will regulate MYC expression in solid tumors that frequently over-express MYC. Here we describe the effects of the highly specific BET inhibitor, I-BET762, on MYC expression in prostate cancer models. I-BET762 potently reduced MYC expression in prostate cancer cell lines and a patient-derived tumor model with subsequent inhibition of cell growth and reduction of tumor burden in vivo. Our data suggests that I-BET762 effects are partially driven by MYC down-regulation and underlines the critical importance of additional mechanisms of I-BET762 induced phenotypes.

Figure 1. Characterization of I-BET762 sensitivity in prostate cancer cell lines

A, Average growth IC₅₀ (gIC₅₀) values observed for I-BET762 in a panel of prostate cancer cell lines obtained from a 6 day growth-death assay (minimum n=2). B, Histograms generated from cell cycle analysis in the indicated cell lines following 6 days treatment with vehicle, 0.5 µM, 1 µM, or 5 µM I-BET762. Data shown was from a single experiment representative of typical results. C, Stacked bar graphs representing the average population of cells in various phases of the cell cycle following treatment with I-BET762 for three days in the indicated cell lines (n=2). D, Western blot analysis of cleaved PARP in the indicated cell lines following three day treatment with a titration of I-BET762.

Figure 2. Expression of various driver oncogenes in prostate cancer cell lines

A, qPCR analysis of basal MYC, AR, ERG, and TMPRSS2-ERG expression in the indicated cell lines. Data were normalized to expression of GAPDH, and are presented as relative expression compared to normal prostate RNA. B, Western blot analysis of basal expression of c-Myc, ERG, AR, and BET proteins in the prostate cancer cell line panel. C, Copy number analysis for MYC in the prostate cancer cell line panel. SK-N-SH and NCI-H82 were included as controls for normal and amplified MYC copy number, respectively.

Figure 3. I-BET762 treatment modulates expression of MYC and c-Myc-driven pathways in prostate cancer cell lines

A, GSEA enrichment plot showing down-regulation of a MYC signature in LNCaP cells treated with 0.5 µM I-BET762 for 24 hours. Normalized enrichment score (NES) and FDR q value are indicated. B, Western blot analysis of c-Myc expression in the indicated cell lines following treatment with a titration of I-BET762 for three days. C, Western blot analysis of c-Myc expression following 3 days treatment with 1µM I-BET762, control siRNA, or MYC siRNA. D, Analysis of cell proliferation in I-BET762 or siRNA-treated LNCaP cells 6 days post-treatment. Data is presented as percent of untreated control cells, and represents the mean +/− SDM for two independent biological replicates.

Figure 4. Persistent MYC expression partially rescues I-BET762-mediated growth effects in LNCaP cells, but has minimal effect in VCaP cells

A, Western blot analysis of c-Myc expression in LNCaP cells (top) and VCaP cells (bottom) overexpressing GFP or MYC following a three day treatment with 1 µM I-BET762. B, Average gIC₅₀ values from a 6 day growth-death assay for LNCaP or VCaP cells overexpressing GFP or MYC (n=3 for LNCaP; n=2 for VCaP). Asterisk indicates statistical significance as determined by t test (p=0.024). C, Stacked bar graphs representing the average population of cells in various phases of the cell cycle following three day treatment with I-BET762 in LNCaP or VCaP cells overexpressing GFP or MYC (n=2 for LNCaP; n=3 for VCaP).

Figure 5. I-BET762 inhibits tumor growth in primary xenograft models with high MYC, AR, and TMPRSS2-ERGexpression

A, qPCR determination of basal expression of the indicated genes in the LUCaP 35CR and LUCaP 145.2 primary xenograft models. Data were normalized to expression of GAPDH, and are presented as relative expression compared to normal prostate RNA. Data represents the mean value ± SEM from three animals. B, qPCR analysis of TMPRSS2-ERG expression as described in A. C, qPCR analysis of gene expression changes induced by I-BET762 treatment in the LuCaP 35CR xenograft model. Expression was analyzed in mice treated daily with vehicle, 8 mg/kg I-BET762, or 25 mg/kg I-BET762 for 36 days. Samples were collected 8 hours after dosing on day 36. Data were normalized to expression of GAPDH, and are presented as fold induction compared to vehicle treated controls. Data represents the mean ± SEM from three animals. Asterisks indicate significant changes as determined by two-tailed, unpaired t test (p< 0.05). D, qPCR analysis of gene expression changes induced by I-BET762 treatment in the LuCaP 145.2 xenograft model. Expression was analyzed in mice treated daily with vehicle, 8 mg/kg I-BET762, or 25 mg/kg I-BET762 for 30 days. Samples were collected 8 hours after dosing on day 30. Data were analyzed and presented as described in C. E, Mean absolute tumor volumes ± SEM for LUCaP 35CR xenografts following treatment with 8 mg/kg or 25 mg/kg I-BET762. Asterisks indicate p < 0.05 as determined by the Mann-Whitney test. TGI for 8 mg/kg was 27% on Day 36 (n= 10; p= 0.44). TGI for 25 mg/kg was 57% on Day 36 (n=10; p =0.006). F, Mean absolute tumor volumes ± SEM for LUCaP 145.2 xenografts following treatment with 8 mg/kg or 25 mg/kg I-BET762. No significant TGI was observed at either dose.