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A.A. Mensah et al.
Discussion
The present study shows that the BET inhibitor OTX015 modulates the expression of miRNAs in DLBCL cells. The regulation may occur directly, due to the bind- ing of BRD4 to the regulatory regions of specific miRNAs, or indirectly as demonstrated for miR-96-5p, a miRNA with important functions in the proliferation and survival of B-cell malignancies.49 The ability of OTX015 to alter miRNA expression demonstrates that the effects of BET inhibition on the transcriptome extend beyond coding genes to comprise also non-coding regions of the genome. miRNAs function by regulating the expression of genes at the transcript level, where they can mediate mRNA degradation, reduce mRNA stability or prevent transla- tion of mRNA into protein.
By analyzing publicly available ChIP sequencing data22,23 in combination with our miRNA profiling data of baseline and BET inhibitor-treated lymphoma cells, we determined that a subset of miRNAs were bound by BRD4 and that this binding decreased after BET inhibitor treatment. For a number of these miRNAs reduced bind- ing of BRD4 after BET inhibition was associated with reduced expression. Our finding that BRD4 directly binds to the regulatory regions of miRNA genes to regulate their expression in lymphomas complements the recent report describing that components of the miRNA process- ing machinery, namely DGCR8 and Drosha, are localized to super-enhancers of miRNAs in a tissue-specific manner and that association of these proteins with super- enhancers is reduced following treatment with the BET inhibitor JQ1.50 Indeed, these observations indicate that the targeted effects of BET inhibition on specific genes in different cellular contexts also likely comprises non-cod- ing transcripts that are specifically modulated in different transformed cell types. With respect to this, we observed that the promoters of two established lymphoma oncomiRNAs, miR-155-5p and miR-92a-1-5p, were bound by BRD4. When lymphoma cells were treated with a BET inhibitor, BRD4 was diminished at these sites and this was associated with downregulation of miRNA expression. miR-155-5p is upregulated in activated B cells and ABC-DLBCL,45,46,51 often because of amplification of its locus.45 Its high expression is associated with poor out- come and resistance to R-CHOP therapy and its knock- down compromises the viability of ABC-DLBCL cells.43,45 Of interest, BET inhibition decreased miR-155-5p expres- sion only in the two ABC-DLBCL cell lines, while the GCB-DLBCL cell line showed upregulation of miR-155- 5p suggesting that different mechanisms may regulate the transcription of this miRNA gene in different DLBCL sub- types.
miR-92a-1-5p is one of the six members of the miR-17- 92 cluster located on chromosome 13q31.3 and is overex- pressed in different lymphoma subtypes including DLBCL.35 It is a transcriptional target of the MYC onco- protein,35 which is itself rapidly and robustly downregu- lated by treatment with a BET inhibitor.15 Downregulation of miR-92a-1-5p was already very pro- nounced after 4 h of BET inhibitor treatment. Additionally, miR-92a-1-5p was the only miRNA com- monly identified by the two different platforms that we utilized for miRNA profiling. The miR-17-92 cluster is involved in activation of the PI3K/AKT/mTOR pathway, lymphoma pathogenesis and chemoresistance.52,53 The
miR-17-92 cluster can acquire super-enhancers during neoplastic transformation50,54 and BRD4 exhibits a prefer- ence for binding at super-enhancers.22 This provides fur- ther support for our observation of direct regulation of the miR-17-92 cluster by BRD4.
miR-21-3p was also downregulated by BET inhibitor treatment. This miRNA is overexpressed in B-cell lym- phomas.40-42 It inhibits translation of the tumor suppressor PTEN43 and knockdown of miR-21 increases the sensitiv- ity of lymphoma cells to CHOP treatment.44
When we performed functional annotation analysis of the OTX015-modulated miRNAs, we identified the same signaling pathways and processes that we had previously identified from gene expression profiles of OTX015-treat- ed DLBCL cells,15 indicating that changes in miRNA expression likely contribute to modulating some of the transcripts and pathways that have been previously iden- tified in cells treated with a BET inhibitor.15,22,23
In lymphoma cells, which overexpress PRMT5, the negative feedback loop comprising miR-96-5p and PRMT5 is usually poised in favor of PRMT5.39 PRMT5 catalyzes the symmetric methylation of arginine residues on histones H3 and H4, giving rise to the repressive epi- genetic marks H3R8me2S and H4R3me2S. Inhibition of PRMT5 expression, either pharmacologically or by the use of antisense oligonucleotides, severely compromises lymphoma cell viability and induces apoptosis.39,49 In the DLBCL cells we treated with OTX015, PRMT5 protein was negligible after 48 h of treatment in the ABC-DLBCL cell line SU-DHL-2, which we previously showed under- goes pronounced apoptosis in response to OTX015.15 DOHH-2 cells, in which we did not observe apoptosis following OTX015 treatment,15 exhibited moderate PRMT5 downregulation. The less marked downregula- tion of PRMT5 protein in DOHH-2 cells treated with OTX015 was in contrast to its pronounced downregula- tion at the transcript level. It was nevertheless associated with a pronounced increase in miR-96-5p levels indicat- ing that, for this cell line, factors other than PRMT5 downregulation may contribute to releasing miR-96-5p from transcriptional repression. Inhibition of PRMT5 has been shown to release miR-96-5p from transcriptional repression in lymphoma cells.39 We observed that BET inhibitor-mediated inhibition of PRMT5 was associated with decreased binding of BRD4 to the 5’ regulatory region of PRMT5 and that this resulted in reduced occu- pancy of PRMT5 at the miR-96-5p promoter. The disrup- tion of the negative feedback loop comprising PRMT5 and miR-96-5p by OTX015 shows how the anti-tumor effects of BET inhibition are further amplified by modu- lation of secondary targets, which might themselves also contribute to further suppress direct targets of BET inhibitors. With respect to this, overexpression of miR- 96-5p downregulates phosphorylated STAT3 (p-STAT3) without affecting levels of total STAT3 in T-cell anaplastic large-cell lymphoma cells.38 We have previously shown that OTX015 treatment decreases p-STAT3 in ABC- DLBCL cells.15 It is therefore possible that in addition to the direct effects of OTX015 on the expression of genes involved in JAK/STAT signaling,15 the overexpression of miR-96-5p contributes to maintaining p-STAT3 repressed.
Our study provides the first evidence of BET inhibitor- mediated modulation of miRNAs in lymphomas. This modulation can occur by inhibiting the interaction of
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haematologica | 2018; 103(12)