Targeting MYC dependence in cancer by inhibiting BET bromodomains

Abstract

The MYC transcription factor is a master regulator of diverse cellular functions and has been long considered a compelling therapeutic target because of its role in a range of human malignancies. However, pharmacologic inhibition of MYC function has proven challenging because of both the diverse mechanisms driving its aberrant expression and the challenge of disrupting protein-DNA interactions. Here, we demonstrate the rapid and potent abrogation of MYC gene transcription by representative small molecule inhibitors of the BET family of chromatin adaptors. MYC transcriptional suppression was observed in the context of the natural, chromosomally translocated, and amplified gene locus. Inhibition of BET bromodomain-promoter interactions and subsequent reduction of MYC transcript and protein levels resulted in G(1) arrest and extensive apoptosis in a variety of leukemia and lymphoma cell lines. Exogenous expression of MYC from an artificial promoter that is resistant to BET regulation significantly protected cells from cell cycle arrest and growth suppression by BET inhibitors. MYC suppression was accompanied by deregulation of the MYC transcriptome, including potent reactivation of the p21 tumor suppressor. Treatment with a BET inhibitor resulted in significant antitumor activity in xenograft models of Burkitt's lymphoma and acute myeloid leukemia. These findings demonstrate that pharmacologic inhibition of MYC is achievable through targeting BET bromodomains. Such inhibitors may have clinical utility given the widespread pathogenetic role of MYC in cancer.

Fig. 1.

Leukemia and lymphoma cell lines are broadly sensitive to BET-bromodomain inhibition. (A) GI₅₀ values of cell lines treated with an active BET inhibitor for 72 h. (B) Dose–response curve of LP-1 cells treated with (+)-JQ1 or (-)-JQ1 for 72 h. (C) Cell cycle profile of LP-1 cells after treatment with DMSO or (+)-JQ1 [625 nM] for 72 h. Data are representative of three independent experiments. (D) Annexin V and PI staining of cells treated with (+)-JQ1 or (-)-JQ1 [5 μM] for 72 h. The percentage of cells that stained positive for annexin V is indicated (red, viable; blue, early apoptosis; pink, late apoptosis). Data are representative of two independent experiments.

Fig. 2.

Gene expression profiling of LP-1 and Raji cells treated with active or inactive BET inhibitors. (A) Top 20 genes down-regulated (blue) and up-regulated (red) by BET-bromodomain inhibition. LP-1 and Raji cells were treated for 4 or 8 h with (+)-JQ1 or (-)-JQ1 at 500 nM. Expression changes for individual time points and cell lines were used to calculate the final expression change score shown in the left column. Detailed data analysis methods, expression data, and differential expression scores can be found in SI Methods. (B and C) GSEA enrichment plots showing the down-regulation of MYC targets in BET-treated cell lines. Genes are ordered by differential expression score. Gene sets are from MSIGDB collections: genes up-regulated in P493-6 (BL) cells induced to express MYC (B); genes with a MYC-MAX binding motif (C). See SI Methods for details.

Fig. 3.

Small molecule BET-bromodomain inhibition suppresses MYC transcription. (A) Quantitative PCR (qPCR) of MYC expression in LP-1 cells treated with DMSO (gray bar) or increasing doses of (+)-JQ1 (blue bars) or (-)-JQ1 for 4 h (black bars). (B) Raji cells were treated with (+)-JQ1 at a final concentration of 1 μM for 2 h. Compound was then washed out of the medium, and samples were taken at indicated time points for qPCR analysis of MYC expression levels. For each time point, MYC expression from DMSO treated sample (gray bar) was set to 100%. (C) Chromatin immunoprecipitation from LP-1 cells treated with DMSO (-) or (+)-JQ1 [500 nM] for 4 h using an antibody against BRD4. qPCR was performed by using primer pairs corresponding to the regions indicated on the x axis. Enrichment relative to no antibody control is indicated. (D) qPCR ChIP from LP-1 cells treated with DMSO (-) or (+)-JQ1 [500 nM] for 4 h using the indicated antibodies. Enrichment relative to no antibody control is indicated. The primer sets used are the following, H3, H4ac, BRD4:-250, and CDK9:TSS.

Fig. 4.

MYC reconstitution significantly protects cells from BET-mediated effects. (A) mRNA expression of MYC and p21 by qPCR upon treatment with (+)-JQ1 in LP-1 cells for the indicated times. mRNA expression is shown relative to DMSO control, where MYC is set to 100% and p21 is set to 1. (B) mRNA expression of MYC or p21 upon (+)-JQ1 treatment for 4 h in LP-1 cells in the absence (MYC off) or the presence (MYC on) of doxycycline. mRNA expression was determined relative to mRNA expression after (-)-JQ1 treatment. (C) LP-1 cells stably transduced with tetracycline-inducible MYC were grown in the absence or presence of doxycycline [250 nM] for 3 d. Cells were then treated with (+)-JQ1 or (-)-JQ1 at 500 nM for 24 h and fixed and stained for FACS analysis of DNA content. The percentage of cells in G₁, S, and G₂/M are indicated. Data are representative of three independent experiments. (D) Quantification of the increase in the percentage of cells in G₁ after (+)-JQ1 treatment in the absence or presence of doxycycline (mean ± SD, n = 3). Values were calculated by subtracting the %G₁ after (-)-JQ1 treatment from the %G₁ after (+)-JQ1 treatment. (E) The relative viability of cells upon treatment of (+)-JQ1 after 4 d in the absence or presence of doxycycline (mean ± SD, n = 3). Values were calculated by determining the ratio of the viability of cells exposed to (+)-JQ1 to the viability of cells exposed to (-)-JQ1 in the absence or presence of doxycycline.

Fig. 5.

BET-bromodomain inhibition decreases tumor load in vivo. (A–C) Mice bearing Raji xenografts were treated with vehicle or with (+)-JQ1 [30 mg/kg (mpk), twice daily, i.p.] for the indicated times. (A) The relative survival of mice in the vehicle group at specific times is indicated under the graph. The dashed line indicates the point in time after which statistical significance cannot be measured accurately because of decreased survival of animals in the control group. Statistical significance for differences in tumor growth rate was determined by using the TTEST method. P value at day 14 = 0.0015. (C) MYC mRNA expression measured by qPCR in s.c. Raji xenografts at the indicated time following a single dose of (+)-JQ1 at 25 mg/kg (mpk). Error bars represent SEM. (B) Kaplan-Meier survival plot. Mice (n = 15 in each group) were killed when tumor volume reached 2,000 mm³. The shown P value was calculated by using the Gehan–Breslow–Wilcoxon test. (D) Mice bearing MV4-11 xenografts were treated with vehicle or with (+)-JQ1 [30 mg/kg (mpk), twice daily, i.p. or 50 mg/kg (mpk) daily, i.p.] for the indicated time. Statistical significance for difference in tumor growth rate was determined by using the TTEST. P value at day 32 were 0.0065 for (+)-JQ1 dosed at 50 mg/kg daily and 0.0008 dosed at 30 mg/kg (mpk) twice a day.