Extended Data Figure 8. JQ1 inhibits ETS (ERG/ETV1) factors that regulate MYC expression in VCaP and LNCaP cells.

a, Genome browser representation of ERG and ETV1 binding events on the MYC distal enhancer⁴². JQ1-treatment in VCaP cells reduces ERG enrichment as shown in two independent ERG ChIP-seq experiments. The y-axis denotes reads per million per base pair (rpm/bp), the x-axis denotes the genomic position. LNCaP ETV1 ChIP-seq data is from Chen et. al. 2013, displaying ETV1 recruitment to the MYC distal enhancer. b, ChIP-PCR validation of loss of ERG recruitment after JQ1-treatment in VCaP cells. Data show mean ± S.D. (n =3) from one of the two independent experiments. c, and d, Knock-down of AR or ETS factor reduces MYC gene expression in VCaP and LNCaP cells. QRT-PCR for AR, ETS and MYC expression in siNT, siAR or siETS transfected cells. Data show mean ± S.D. (n =3) from one of the two independent experiments. e, A cartoon illustrating the mechanism of MYC loss by JQ1 in AR positive VCaP and LNCaP cells. f, Anti-androgens but not JQ1 de-repress MYC expression in prostate cancer cells. Genome browser representation of AR and RNA PolII binding events within the MYC gene locus. The y-axis denotes reads per million per base pair (rpm/bp), the x-axis denotes the genomic position. Note the AR recruitment to the same distal enhancer which is occupied by ERG (see Extended Data Fig. 8a), implicating a competition between AR and ETS factor to bind to this enhancer region to regulate MYC gene expression. g, Heat map showing the MYC expression values from VCaP microarray gene expression data. h, Anti-androgen restores DHT-repressed MYC expression in VCaP cells. QRT-PCR of MYC in VCaP cells treated with vehicle, DHT (10nM), DHT+JQ1 (500nM), DHT+MDV3100 (10μM) or DHT+Bicalutamide (25μM). Inability of JQ1 to de-repress MYC in this setting could be explained by the fact that both AR and ERG is de-recruited from MYC distal enhancer leading to net loss of MYC expression. i, MDV3100 and not JQ1 restores DHT-repressed MYC protein levels in VCaP cells. Immunoblot of MYC protein in VCaP cells pre-treated with vehicle, MDV3100 (10μM) or JQ1 (500nM) for 4hrs followed by DHT (10nM) for 20hrs. Data show mean ± S.D.(n =3) from one of the two independent experiments. NS, not significant; *P ≤ 0.01; **P ≤ 0.001; **P ≤ 0.0001 by two-tailed Student's t-test.