Targeting Bromodomain and External Domain Epigenetic Reader Protein As Effective Strategy for Double-Hit and Triple-Hit B-Cell Lymphoma

Approximately 12% of diffuse large B cell lymphoma (DLBCL) are double-hit lymphomas (DHL) with translocations in both MYC (MYC/8q24) and BCL2 (14;18) (q32; q21) and/or BCL6 (3q27) and prognosis is generally poor. Triple hit lymphomas (THL) are a subset of DHL exhibiting simultaneous gene translocations of MYC, BCL2 and BCL6, and demonstrate aggressive behavior, worse than that of DHL. Currently, there are no guidelines for the treatment of this group of patients, and disease remains challenging to treat. Bromodomain and external domain (BET) proteins are known as "epigenetic readers" and are enriched at the regions regulating transcription of oncogenes and many BET inhibitors (BETi) are now being clinically evaluated in several cancers.

The overall goal of this study was to investigate the effect of BET inhibition through pharmacological inhibitors on single hit, DHL and THL lymphomas. We have performed extensive cytogenetic analysis on 14 human B cell lymphoma cell lines to identify MYC, BCL2 and BCL6 translocations. We identified DOHH2, OCILy1, OCILy10 as DHL, while only VAL represents THL. Raji was identified as single MYC translocation (SHL) and U2932 as WT MYC. Proliferation rate for each cell line was assessed by thymidine incorporation assays in the presence or absence of BET inhibition through pharmacological inhibitors JQ1, IBET762 or OTX015 in WT, SHL, DHL and THL B cells. BET inhibition through each BET inhibitors used individually led to significant (p=0.001) inhibition of cell proliferation in DHL and THL cells or WT- and SH- MYC expressing cells. As expected, BET inhibition led to almost complete inhibition of MYC mRNA and MYC protein in multiple lymphoma cell lines regardless of cytogenetic features. Overexpression of MYC partially rescued the effects of BET inhibition in these cells. Chromatin immunoprecipitation assay was performed to further examine the MYC regulatory mechanisms by BET inhibition. JQ1 treatment reduced BRD4 occupancy on MYC promoter but had no effect on BRD4 occupancy at the promoter region of BCL6. Overall these findings suggest that BET inhibition may suppress proliferation of DHL and THL cells via disruption of transcriptional network led by oncogenic MYC. Interestingly, the effect of BET inhibition on cell survival was modest in all MYC expressing cell lines regardless of their cytogenetic characteristics. Histone acetylation marks are major binding sites for BET proteins. We sought to assess the effect of combining BET inhibitors with histone deacetylase inhibitor vorinostat on proliferation and survival in WT, DHL and THL cells. Combination of sub-lethal doses of IBET and vorinostat decreased proliferation of WT, DHL and THL cells to a greater extent as compared to either drug alone (p<0.0001). IBET/vorinostat combination had no greater effect than IBET or SAHA alone on cell survival or signaling proteins (MCl-1, BCL2 and BIM) in any of the MYC expressing cell lines. These results suggest that combination of BET and/or HDAC pathway can greatly influence cell proliferation but had very limited impact on survival of MYC expressing WT, DHL or THL cells in-vitro. We observed that BET inhibition attenuated BCL6 expression but had no effect on BCL2 expression in DHL and THL expressing B cells. Next we tested the combination of JQ1 and IBET with a BCL2 inhibitor ABT199 (ABT) and assessed survival in THL and DHL cells. ABT alone decreased cell survival in a dose dependent manner, while co-treatment of IBET or JQ1 with ABT significantly suppressed survival as compared to ABT alone (p<0.001). We also evaluated the immunomodulatory activity of the BET inhibitors on the immune checkpoint PD-L1 and CD47, an anti-phagocytic ligand expressed on tumor cells. CD47 surface expression was observed in both DHL and THL cells, however constitutive PD-L1 was only expressed on THL cells; Both CD47 and PD-L1 expression decreased with JQ1 and IBET treatment (p<0.03). These data suggest that BET inhibitors may play an immunomodulatory role in suppressing MYC in the DHL and THL lymphomas.

This study shows for the first time the role of BET epigenetic reader protein in DHL and THL aggressive B-cell lymphoma. Using several pharmacological inhibitors, we provide comprehensive mechanistic studies of its action to provide a rationale for the use of BET inhibitor therapy alone or in combination with BCL2 or checkpoint inhibitors for patients with double-hit and triple-hit lymphoma.