Abstract B76: Evaluation of the novel BET Bromodomain inhibitor BAY 1238097 in lymphoma models identifies EZH2 and MYD88 mutations as potential biomarkers and the bases for combinations
Authors: Bernasconi Elena , Gaudio Eugenio , Tarantelli Chiara , Cascione Luciano , Kwee Ivo , Rinaldi Andrea , Mensah Afua A. , Stathis Anastasios , Ocker Matthias , Zucca Emanuele , Haendler Bernard , Bertoni Francesco 
Published: May 2016

Abstract
Introduction. Lymphomas present frequent alterations of proteins involved in the epigenetic regulation of gene expression. BET Bromodomain proteins are epigenetic readers contributing to gene transcription regulation. Here, we present preclinical results on the novel BET Bromodomain inhibitor BAY 1238097, currently in phase I study (NCT02369029), alone and in combination in lymphoma cell lines.
Methods. Cell lines derived from diffuse large B-cell lymphoma (DLBCL, 27), mantle cell lymphoma (MCL, 10), splenic marginal zone lymphoma (3), anaplastic large T cell lymphoma (9), pro-lymphocytic leukemia and primary mediastinal large cell lymphoma (1 each) were exposed to increasing concentrations of BAY 1238097 for 72 h followed by MTT assay. Sensitivity was correlated with baseline gene expression profiles and genetic features. Synergy was assessed with the Chou-Talalay combination index (CI): &lt;0.9, synergism; 0.9-1.1 additive effect; &gt;1.2, no benefit.
Results. BAY 1238097 had a median IC50 of 208 nM (95%C.I., 157-260). Activity was higher in B- than in T-cell lymphomas (P&lt;0.001), while no differences were observed among B-cell tumor types or DLBCL ABC/GCB subtypes. Anti-tumor activity was mainly cytostatic, as evidenced by LC50 values higher than 1 μM in 43/51 cell lines, with G1-S block (3/3 cell lines treated with BAY 1238097, 500nM/72h). However, LC50 values &lt; 1 μM were obtained in 7/51 (14%) cell lines (all DLBCL/MCL) and apoptosis was confirmed in all of them by Annexin-V/7-AAD staining. Mut EZH2 was associated with higher sensitivity in all cell lines (P&lt;0.05) and in GCB-DLBCL (P&lt;0.05). Also, an EZH2 signature from follicular lymphomas with or without mutEZH2 (Guo et al., CCR 2014) was associated with higher sensitivity in GCB-DLBCL (NES = 1.6, P&lt;0.01). Thus, we evaluated the effect of BAY 1238097 on EZH2 activity. The compound (500 nM, 72 h) down-regulated EZH2 protein levels and H3K27me3 in 4/4 DLBCL (2 wt, 2 mut EZH2). We then analyzed publicly available ChIP-Seq data for HBL1 DLBCL treated with the BET bromodomain inhibitor JQ1 (500 nM, 3 h; Ceribelli et al, PNAS 2014): the treatment reduced BRD4 binding to the EZH2 upstream regulatory region, indicating a direct effect of BET Bromodomain inhibitors on the regulation of the EZH2 mRNA. Finally, we assessed the combination of BAY 1238097 with the EZH2-inhibitor DZNep for 72 h in 8 GCB-DLBCL. The combination was synergistic or additive in 4/5 mut EZH2 GCB-DLBCL (WSU-DLCL2, CI = 0.7; KARPAS422, 0.7; SU-DHL6, 1; SU-DHL4, 1.1), while it did not present any benefit in 3/3 GCB-DLBCL cell lines with wt EZH2 (Toledo, CI = 1.3; DOHH2, CI = 1.6; Farage, CI = 3.1).
In ABC-DLBCL L265P-MYD88 (P = 0.05) and high expression of JAK/STAT and IFN signaling genes were positively associated with sensitivity. Since MYD88 is a known target of BET Bromodomain inhibitors (Boi et al, CCR 2015), we assessed the combination of BAY 1238097 with the BTK-inhibitor ibrutinib for 72 h and synergism was observed in 2/2 ABC-DLBCL cell lines harboring L265P-MYD88 (OCI-Ly10, CI = 0.8; TMD8, 0.6). No benefit was observed in 2/2 ABC-DLBCL with wt MYD88 (SU-DHL2, CI = 1.7; U2932, 1.4).
Conclusions. BAY 1238097 was active in preclinical models of mature lymphomas. Mutations in EZH2 and MYD88 appeared as potential biomarkers and suggested rational combinations to be further explored.
Citation Format: Elena Bernasconi, Eugenio Gaudio, Chiara Tarantelli, Luciano Cascione, Ivo Kwee, Andrea Rinaldi, Afua A. Mensah, Anastasios Stathis, Matthias Ocker, Emanuele Zucca, Bernard Haendler, Francesco Bertoni. Evaluation of the novel BET Bromodomain inhibitor BAY 1238097 in lymphoma models identifies EZH2 and MYD88 mutations as potential biomarkers and the bases for combinations. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B76.