ingest cdrApp 2019-01-04T15:00:11.808Z 2a9effba-8beb-4434-9ac1-3dd15c1ac330 modifyDatastreamByValue RELS-EXT fedoraAdmin 2019-01-04T15:01:13.011Z Setting exclusive relation addDatastream MD_TECHNICAL fedoraAdmin 2019-01-04T15:01:28.190Z Adding technical metadata derived by FITS addDatastream MD_FULL_TEXT fedoraAdmin 2019-01-04T15:01:55.157Z Adding full text metadata extracted by Apache Tika modifyDatastreamByValue RELS-EXT fedoraAdmin 2019-01-04T15:02:20.098Z Setting exclusive relation modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2019-01-22T16:04:15.438Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2019-03-20T13:42:31.491Z Samantha Bevill Author Department of Pharmacology School of Medicine TRANSCRIPTIONAL ADAPTATION TO TARGETED INHIBITORS VIA BET BROMODOMAIN PROTEINS IN TRIPLE-NEGATIVE BREAST CANCER 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. Winter 2018 2018 Pharmacology Oncology Molecular biology BET bromodomain, breast cancer, drug resistance, trametinib eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Pharmacology Gary Johnson Thesis advisor Channing Der Thesis advisor Michael Emanuele Thesis advisor Carey Anders Thesis advisor Jill Dowen Thesis advisor text Samantha Bevill Creator Department of Pharmacology School of Medicine TRANSCRIPTIONAL ADAPTATION TO TARGETED INHIBITORS VIA BET BROMODOMAIN PROTEINS IN TRIPLE-NEGATIVE BREAST CANCER 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. 2018 2018-12 Pharmacology Oncology Molecular biology BET bromodomain; breast cancer; drug resistance; trametinib eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Pharmacology Gary Johnson Thesis advisor Channing Der Thesis advisor Michael Emanuele Thesis advisor Carey Anders Thesis advisor Jill Dowen Thesis advisor text Samantha Bevill Creator Department of Pharmacology School of Medicine 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. 2018 2018-12 Pharmacology Oncology Molecular biology BET bromodomain; breast cancer; drug resistance; trametinib eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Gary Johnson Thesis advisor Channing Der Thesis advisor Michael Emanuele Thesis advisor Carey Anders Thesis advisor Jill Dowen Thesis advisor text Bevill_unc_0153D_18234.pdf uuid:2ca5705a-c398-4f76-a375-11b315596ac9 2020-12-31T00:00:00 2018-12-01T17:56:18Z proquest application/pdf 18151567