The Combination of a BET Inhibitor (GS-5829) and a BTK Inhibitor (GS-4059) Potentiates DLBCL Cell Line Cell Death and Reduces Expression of MYC, IL-10, and IL-6 in Vitro
Authors: Bates Jamie , Kusam Saritha , Tannheimer Stacey , Chan Julie , Li Yvonne , Breckenridge David , Tumas Daniel 
Published: October 2019

Abstract
Introduction: GS-5829 is a potent and selective inhibitor of thebromodomain and extra-terminal (BET) family of proteins. BET proteins regulate the transcription of key oncogenes including MYC resulting in cancer cell growth (Yang Z. et. al.Mol CellBiol 2008; LeRoy G. et al.Mol Cell 2008). DLBCL is a diverse and genetically heterogeneous subtype of non-Hodgkin's lymphoma (NHL) (Lenz G. et. al. NEngl J Med 2010). The combination of a BET inhibitor, CPI203, and aBruton's tyrosine kinase (BTK) inhibitor was reported to have synergistic efficacy in vitro and in an in vivo xenograft model with the TMD8 cell line derived from the ABC subtype of DLBCL (Ceribelli M. et. al. Proc NatlAcadSci USA 2014). We tested the combination of the BET inhibitor, GS-5829, and the BTK inhibitor, GS-4059, to inhibit cell growth and reduce IL-6, IL-10, and MYC transcripts and protein in the ABC-DLBCL cell line, TMD8, in vitro.
Methods: Cells were treated with GS-4059 alone or with GS-5829 using clinically achievable concentrations. The activity in the TMD8 cell line was evaluated after 72 hours and cell growth was assessed using CellTiterGlo. Synergy was determined by BLISS score analysis (Greco W. et. al. Pharmacol Rev 1995). The EC50 value for each compound was determined using GraphPad Prism software by fitting the data to a 4-parameter variable slope model. IL-6, IL-10, and MYC transcripts were quantified from cell lysates using NanoString analyses. MYC protein was quantified in cell lysates using ProteinSimple quantification and IL-6 and IL-10 protein levels were quantified in supernatants using Luminex cytokine analysis (Bio-Plex Human Cytokine Assay).
Results: The EC50 for growth inhibition was 25 nM for both the BET inhibitor, GS-5829, and for the BTK inhibitor GS-4059 alone. The combination of the 2 inhibitors resulted in a mean BLISS score of 160. Addition of 5 - 10 nM of the BTK inhibitor resulted in a 2-3 fold decrease in the EC50 of the BET inhibitor. The combination reduced MYC, IL-6, and IL-10 transcripts and protein, with low concentrations causing significant reduction of IL-6 and IL-10 protein levels compared to either single agent alone (p <0.05; 1-way ANOVA).
Conclusion: The data suggest that combination of GS-4059 and GS-5829 could theoretically have increased activity in patients with the ABC subtype of DLBCL.
Figure The Addition of a BTK Inhibitor Decreases the Viability EC50 of GS-5829 in the ABC-DLBCL Cell Line, TMD8, In Vitro Figure. The Addition of a BTK Inhibitor Decreases the Viability EC50 of GS-5829 in the ABC-DLBCL Cell Line, TMD8, In Vitro

Disclosures
Bates: Gilead Sciences: Employment, Equity Ownership. Kusam:Gilead Sciences: Employment, Equity Ownership. Tannheimer:Gilead Sciences: Employment. Chan:Gilead Sciences: Employment, Equity Ownership. Li:Gilead Sciences: Employment, Equity Ownership. Breckenridge:Gilead Sciences: Employment, Equity Ownership. Tumas:Gilead Sciences: Employment, Equity Ownership.