Abstract
Background: Bromodomain and extra terminal (BET) family members are being targeted by small molecules in early phase clinical trials for hematopoietic malignancies. These compounds act by binding to the bromodomain of BET family members to block their association with acetylated lysines that are largely on the N-terminal tails of histones. While tool compounds such as JQ1 have been used to show that BET inhibitors impair the transcription of MYC, BCL2 and other oncogenes, only select cell lines are sensitive to these compounds. An in depth analysis of BET family members and the action of these compounds has been complicated by a lack of tools to dissect the effects of these factors on transcription rates rather than steady-state cytoplasmic mRNA pools.
Methods: We used precision nuclear run-on transcription sequencing (PRO-Seq) to create high-resolution genomic maps of the locations of all active RNA polymerases (including Pol I, Pol II, and Pol III) before and after treatment with BET inhibitors (BETi). Short treatment times with BETi were used to identify the immediate effects of inhibiting these factors on transcription before compensatory changes occurred. We used Kasumi-1 and SKNO-1 t(8;21) containing AML cells as model systems, as these cells are exceptionally sensitive to these compounds.
Results: We found that a 1 hr treatment with BETi caused RNA polymerase pausing within 20-50 bp downstream of the transcription start site in over 1700 genes, including KIT, which is mutated in up to 1/3 of t(8;21) AML. PRO-Seq also identified a 34-kb long eRNA transcribed from what appears to be a group of enhancers 3' to KIT, which was repressed by BETi. KIT protein was dramatically reduced by BETi, suggesting that loss of mutated/amplified KIT contributes to the exceptional response of t(8;21) AML to these drugs. Conversely, over 200 genes showed enhanced transcription in the presence of BETi including MCL1. In addition, BETi repressed the MCL1-targeting microRNAs, MIR29C and MIR29B2. MCL1 protein expression was up-regulated throughout the treatment, suggesting a potential mechanism of resistance to BETi-induced cell death. While comparing the transcriptional effects of different inhibitors of BET family members, INCB054329 was 2-4 fold more potent than JQ1 at inhibiting the growth of Kasumi-1 and SKNO-1 cells
Conclusions: Bromodomain inhibition leads to up-regulation of MCL1, which may impair cell death. In addition, BETi leads to dramatic reduction of KIT transcription by INCB054329, and may lend insight to the use of BETi in the treatment of t(8;21) AML, or other AML that contain mutated or amplified KIT.
Liu:Incyte: Employment. Scherle:Incyte: Employment. Huber:Incyte Corporation: Employment, Equity Ownership. Savona:Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Astex: Research Funding. Hiebert:Incyte Corporation: Research Funding.
Author notes
Asterisk with author names denotes non-ASH members.
