Abstract
Bromodomain and extra-terminal domain (BET) family inhibitors offer an 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), as these polymerases are exceptionally sensitive to BET inhibitors. PRO-seq identified over 1,400 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, while 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 upregulated, 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.
Highlights
The bromodomain and extra-terminal domain (BET) proteins consist of four family members including BRD2, BRD3, BRD4, and BRDT (Wu and Chiang, 2007)
BET proteins are typically associated with enhancers and bind to positive transcription elongation factor b (P-TEFb, which contains cyclin T and CDK9), which is critical for the release of promoter-proximal paused RNA polymerases into productive elongation (Chapuy et al, 2013; Liao et al, 1995; Loven et al, 2013; Marshall and Price, 1995; Peng et al, 1998; Yang et al, 2005)
BET Inhibitors Cause Promoter-Proximal Pausing of RNA Polymerases Early studies identified acute myeloid leukemia (AML) as especially sensitive to inhibitors of BET family members, and the t(8;21) cell line Kasumi-1 appeared to be the most sensitive cell line (Zuber et al, 2011). We extended these results to the t(8;21)-containing SKNO-1 cell line that requires granulocyte-macrophage colony stimulating factor (GM-CSF) for growth (Matozaki et al, 1995) and found that Kasumi-1 and SKNO-1 cells were more sensitive than MOLM13 and MV4-11 when using alamarBlue assays to assess cell metabolism as a surrogate for cell proliferation
Summary
The bromodomain and extra-terminal domain (BET) proteins consist of four family members including BRD2, BRD3, BRD4, and BRDT (Wu and Chiang, 2007). The binding of BRD4 appears to activate P-TEFb by releasing it from the inhibitory HEXIM17SK complex (Bartholomeeusen et al, 2012; Jang et al, 2005; Liu et al, 2014) This stimulates CDK9-dependent phosphorylation of the C-terminal domain of RNA polymerase II (RNAPII) as well as negative elongation complexes, DSIF and NELF, which causes dissociation of NELF and switches DSIF into a positive elongation factor, to trigger RNAPII elongation (Wada et al, 1998a, 1998b; Yamaguchi et al, 1999). BET inhibitors show efficacy in preclinical models of acute myeloid leukemia (AML), multiple myeloma, and certain types of lymphoma as well as other cancer types (Chapuy et al, 2013; Dawson et al, 2011; Delmore et al, 2011; Feng et al, 2014; Filippakopoulos et al, 2010; Lockwood et al, 2012; Ott et al, 2012; Zuber et al, 2011)
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