Genome-Wide Analysis of BRD4-Targets Reveals the Therapeutic Relevance of Simultaneous Targeting of BCR Pathway and IKZF-MYC Axis in Mantle Cell Lymphoma
Authors: Kuroda Junya , Tsukamoto Taku , Nakahata Shingo , Morishita Kazuhiro , Sato Ryuichi , Kanai Akinori , Inaba Toshiya , Nakano Masakazu , Tashiro Kei , Taniwaki Masafumi 
Published: October 2019

Abstract
Mantle cell lymphoma (MCL) has been mostly incurable, and there is an urgent need to identify targetable molecules for development of a more effective treatment strategy. Bromodomain and extraterminal domain (BET) proteins associate with acetylated histones and facilitate transcription of target genes, and bromodomain-containing 4 (BRD4), a member of BET proteins, recruits the P-TEFb complex to genomic lesions in chromatin and thereby activates RNA Pol II at specific promoter sites of target genes. In addition, super-enhancers have been recognized as regulatory regions with a high level of acetylated histones, mediator complexes and BRD4, and super-enhancers in cancer cells are enriched at oncogenes. Recent studies have shown that BRD4 promotes expression of pivotal molecules in disease development, maintenance and progression in various cancers, including lymphoma. Given, we in this study examined the effect of BRD4 inhibition on human MCL-derived cell lines, Jeko-1, JVM-2, MINO and Z138, and performed broad screening of BRD4-regulated molecules using genome-wide approaches to identify therapeutic targets for MCL. As the results, treatment with a BRD4 inhibitor I-BET151 for 72 h showed a dose-dependent inhibitory effect on cell proliferation in all four cell lines, with half maximal inhibitory concentrations (IC50s) of 15.6 nM, 3.6 nM, 2.6 nM and 3.0 nM in Jeko-1 cells, JVM2 cells, MINO cells and Z138 cells, respectively, which was accompanied by G1/S cell cycle arrest and the induction of apoptosis. Next, we performed comprehensive gene expression profile (GEP) analysis for JVM2 and Z138 cells with or without I-BET151 treatment, and BRD4 chromatin immunoprecipitation sequencing (ChIP-Seq) in JVM2 cells treated with 10 nM I-BET151 or DMSO. Accordingly, GEP analyses revealed that more than 600 genes were commonly upregulated by more than 1.5-fold and downregulated by less than 0.67-fold, respectively, in JVM2 and Z138 cells treated by I-BET151, while ChIP-Seq showed that 7988 BRD4-binding regions were dysregulated by I-BET151, with most of these sites in enhancer regions, and 547 BRD4-binding regions were characterized as super-enhancers. Integrated analysis using the Reactome Pathway Database and the results of GEP and ChIP-Seq showed that a series of genes involved in the B cell receptor (BCR) signaling pathway and IKZF-MYC axis are regulated by BRD4 in MCL cells. To confirm whether each BRD4 target contributes to survival and proliferation of MCL cells, we focused on several candidate targets: the BCR pathway, IKZF and MYB. However, ibrutinib, a Bruton kinase inhibitor, suppressed cell growth in only two of the four cell lines (MINO and JVM2) in a dose-dependent manner, while lenalidomide, an inhibitor of the IKZF family, did not affect cell survival, despite its potency in decreasing IKZF1 and IKZF3 proteins. MYB silencing using shMYB did not decrease cell proliferation in any of the four MCL cell lines. In conclusion, our study disclosed that BRD4 regulates transcription of multiple genes by binding to enhancer region, partly involving super-enhancers and multiple known pathways, such as BCR signaling and the IKZF-MYC axis, which play essential roles in survival of MCL cells. While the efficacy of single targeting of BCR-signaling, IKZF, or MYB was limited, I-BET151 concomitantly inactivated the BCR pathway and IKZF and had a high growth inhibitory efficacy in MCL cells. These results suggest that simultaneous targeting of multiple molecules involved in the BCR pathway and IKZF-MYC axis may overcome resistance to ibrutinib and/or lenalidomide in MCL, and that BRD4 inhibitors are promising candidates for MCL treatment.

Disclosures
Kuroda: Chugai Pharma: Honoraria, Research Funding. Taniwaki:Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.,: Research Funding; Astellas Pharma Inc,: Research Funding.