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  • March 20, 2019
  • Bevill, Samantha
    • Affiliation: School of Medicine, Department of Pharmacology
  • Targeted kinase inhibitors have displayed limited efficacy in treating breast cancer due to the ability of tumor cells to upregulate bypass signaling networks in response to treatment. Triple-negative breast cancers (TNBCs) often present with dysregulation of the BRaf-MEK-ERK pathway, making them sensitive to MEK inhibitor (MEKi) treatment. Despite initial clinical responses, drug resistance often develops involving non-genomic adaptive bypass mechanisms. Inhibition of MEK1/2 by trametinib in TNBC patients induced dramatic transcriptional responses, including upregulation of receptor tyrosine kinases (RTKs) when comparing tumor samples before and after one week of treatment. In preclinical models, MEK inhibition induced genome-wide enhancer formation involving the seeding of BRD4, MED1, H3K27 acetylation and p300 that drove transcriptional adaptation. Inhibition of P-TEFb associated proteins arrested enhancer seeding and RTK upregulation. BRD4 bromodomain inhibitors or RNAi knockdown of BRD4 overcame trametinib resistance, producing sustained growth inhibition in cells, xenografts and syngeneic mouse TNBC models. These data highlight pharmacological targeting of P-TEFb members, including BET bromodomains, in conjunction with MEK inhibition as an effective strategy to durably inhibit epigenomic remodeling required for adaptive resistance. The ability of BET bromodomain inhibitors to block the adaptive response of TNBC to MEKi made us question whether BET bromodomain inhibitors could block adaptation to targeted inhibition of additional kinases or signaling pathways. Screening of an inhibitor library targeting kinases and epigenetic regulators identified a series of molecules which displayed anti-proliferative synergy with BET bromodomain inhibitors (JQ1, OTX015) in TNBC. GSK2801, an inhibitor of BAZ2A/B bromodomains, of the imitation switch chromatin remodeling complexes, and BRD9, of the SWI/SNF complex, demonstrated unique synergy independent of BRD4 control of P-TEFb-mediated pause-release of RNA polymerase II. GSK2801, or RNAi knockdown of BAZ2A/B, in combination with JQ1 selectively displaced BRD2 at promoters/enhancers of ETS-regulated genes. Additional displacement of BRD2 from ribosomal DNA in the nucleolus coincided with decreased 45S rRNA, revealing a function of BRD2 in regulating RNA polymerase I transcription. In 2D cultures, enhanced displacement of BRD2 from chromatin by combination drug treatment induced senescence. In spheroid cultures, combination treatment induced cleaved caspase-3 characteristic of apoptosis in tumor cells but not co-cultured mammary fibroblasts. Thus, GSK2801 blocks BRD2-driven transcription in combination with BET inhibition and induces apoptosis of TNBC. Cumulatively, the data presented in this thesis provides a series of synergistic drug combinations which effectively inhibit growth and survival of TNBC in combination with targeted inhibitors via inhibition of P-TEFb transcriptional elongation by BRD4, or regulation of ETS target genes and rRNA transcription by BRD2.
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Rights statement
  • In Copyright
  • Emanuele, Michael
  • Johnson, Gary
  • Dowen, Jill
  • Der, Channing
  • Anders, Carey
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2018

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