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Vassiliki Poulaki, Nicholas Mitsiades, Warren Fiskus; Bromodomain (BRD) inhibition as a novel strategy to inhibit the Microphthalmia-associated transcription factor (MITF) axis in uveal melanoma (UM). Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5337.
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© ARVO (1962-2015); The Authors (2016-present)
UM is universally lethal when metastatic, creating an unmet need for novel, effective, targeted systemic therapies. MITF is a critical oncogenic transcription factor in UM, yet no targeted therapies are available to inhibit it. BRDs are epigenetic readers that recognize acetylated lysine on chromatin proteins and regulate gene transcription. Lysine 27-specific acetylation of Histone 3 (H3K27ac) is associated with enhancer sites on chromatin and active transcription.
We performed Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) analysis for BRD4, H3K27ac, MITF and RNA Polymerase II (RNAP2) in UM cells. We also examined the transcriptomic and proteomic effects of BRD4 siRNA and of the BRD inhibitor JQ1 alone and in combination with the PKC inhibitors bisindolylmaleimide I (BIM) or AEB071 against a panel of five UM cell lines in vitro and in a UM xenograft model.
Integrated ChIP-Seq analysis demonstrated strong genome-wide co-localization of BRD4, H3K27ac, MITF and RNAP2, suggesting that they cooperate to drive MITF-dependent transcription. Treatment of UM cells with JQ1 abolished the recruitment of BRD4 to the c-Myc and MITF genes, and suppressed the expression of c-Myc and MITF mRNAs and proteins, as well as of MITF target genes, in multiple UM cell lines. These effects were also encountered upon treatment with BRD4 siRNA. Global gene expression analysis revealed that JQ1 induces a transcriptional signature that is associated with decreased MITF activity, decreased metastatic potential and improved patient survival when applied to publicly available UM patient datasets. JQ1 increased expression of p21, p27, BCL2L11 and cleaved PARP proteins in UM cells. These effects were enhanced by the combination of JQ1 with BIM or AEB071, which led to synergistic loss of cell viability (by isobologram analysis; CI < 1.0). JQ1 significantly inhibited the growth of UM xenografts in SCID/Beige mice (p < 0.01).
BRD4 is a central regulator of MITF-driven oncogenic signaling in UM. BRD inhibition silences c-myc and MITF expression and function, inhibits proliferation and induces apoptosis, holding promise for the treatment of UM, either as monotherapy or in combination with PKC inhibitors.
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