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High Resolution Mapping of RNA Polymerases Identifies Mechanisms of Sensitivity and Resistance to BET Inhibitors in t(8;21) AML

ABSTRACT: Bromodomain and extra-terminal domain (BET) family inhibitors offer a new approach to treating hematological malignancies. We used precision nuclear run-on transcription sequencing (PRO-seq) to create high-resolution maps of active RNA polymerases across the genome in t(8;21) acute myeloid leukemia (AML) that are exceptionally sensitive to BET inhibitors. PRO-seq identified over 1400 genes showing impaired release of promoter-proximal paused RNA polymerases, including the stem cell factor receptor tyrosine kinase KIT that is mutated in t(8;21) AML. PRO-seq also identified an enhancer 3’ to KIT. Chromosome conformation capture confirmed contacts between this enhancer and the KIT promoter and CRISPRi-mediated repression of this enhancer impaired cell growth. PRO-seq also identified microRNAs, including MIR29C and MIR29B2 that target the anti-apoptotic factor MCL1 and were repressed by BET inhibitors. MCL1 protein was up-regulated, and inhibition of BET proteins sensitized t(8:21)-containing cells to MCL1 inhibition, suggesting a potential mechanism of resistance to BET inhibitor-induced cell death.

ORGANISM(S): Homo sapiens

PROVIDER: GSE83660 | GEO | 2016/07/15

SECONDARY ACCESSION(S): PRJNA326664

REPOSITORIES: GEO

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Project description:Following the discovery of BRD4 as a non-oncogene addiction target in acute myeloid leukemia (AML), BET inhibitors are being explored as promising therapeutic avenue in numerous cancers. While clinical trials have reported single-agent activity in advanced hematologic malignancies, mechanisms determining the response to BET inhibition remain poorly understood. To identify factors involved in primary and acquired BET resistance in leukemia, we performed a chromatin-focused shRNAmir screen in a sensitive MLL/AF9; NrasG12D‑driven AML model, and investigated dynamic transcriptional profiles in sensitive and resistant murine and human leukemias. Our screen reveals that suppression of the PRC2 complex, contrary to effects in other contexts, promotes BET resistance in AML. PRC2 suppression does not directly affect the regulation of Brd4-dependent transcripts, but facilitates the remodeling of regulatory pathways that restore the transcription of key targets such as Myc. Similarly, while BET inhibition triggers acute MYC repression in human leukemias regardless of their sensitivity, resistant leukemias are uniformly characterized by their ability to rapidly restore MYC transcription. This process involves the activation and recruitment of WNT signaling components, which compensate for the loss of BRD4 and drive resistance in various cancer models. Dynamic ChIP- and STARR-seq enhancer profiles reveal that BET-resistant states are characterized by remodeled regulatory landscapes, involving the activation of a focal MYC enhancer that recruits WNT machinery in response to BET inhibition. Together, our results identify and validate WNT signaling as a driver and candidate biomarker of primary and acquired BET resistance in leukemia, and implicate the rewiring of transcriptional programs as an important mechanism promoting resistance to BET inhibitors and, potentially, other chromatin-targeted therapies. RNA-Seq of DMSO- or JQ1-treated cancer cell lines; ChIP-seq for H3K36me3 and H3K27me3 in a leukemia cell line treated either with DMSO or JQ1, ChIP-seq for H3K27ac in resistant and sensitive mouse and human leukemia. Functional enhancer mapping (STARR-seq) in K-562 treated either with DMSO or JQ1.

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High Resolution Mapping of RNA Polymerases Identifies Mechanisms of Sensitivity and Resistance to BET Inhibitors in t(8;21) AML

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