Entinostat, a Novel Histone Deacetylase (HiDAC) Inhibitor Enhances the Anti-Tumor Activity of Bortezomib (BTZ) in Rituximab-Chemotherapy Sensitive and Resistant Lymphoma Cell Lines,
Authors: Mavis Cory , Frys Sarah , Gu Juan , Gibbs John , Czuczman Myron S. , Hernandez-Ilizaliturri Francisco J. 
Published: October 2019

Abstract 3734

Deacetylases (DACs) are enzymes that remove the acetyl groups from target proteins [histones (class I) and non-histone proteins (class II)], leading to regulation of gene transcription and other cellular processes. Entinostat (MS-275) is a novel and potent DAC class I inhibitor undergoing pre-clinical and clinical testing. In order to better characterize the role of DAC inhibitors in the treatment of refractory/resistant (r/r) B-cell lymphoma, we studied the anti-tumor activity of entinostat as a single agent or in combination with the proteasome inhibitor bortezomib (BTZ) against a panel of rituximab-[chemotherapy]-sensitive cell lines (RSCL), rituximab-[chemotherapy]-resistant cell lines (RRCL), and primary lymphoma cells isolated from patients with treatment-naïve or r/r B-cell lymphoma. In addition, we characterized the mechanisms responsible for entinostat's anti-tumor activity. Non-Hodgkin lymphoma (NHL) cell lines were exposed to escalating doses of entinostat (0.1 to 20uM) +/− BTZ (1–10nM). Changes in mitochondrial potential and ATP synthesis were determined by alamar blue reduction and cell titer glo luminescent assays, respectively. Changes in cell cycle were determined by flow cytometric analysis. Subsequently, protein lysates were isolated from entinostat +/− BTZ exposed cells and changes in members of Bcl-2 and cell cycle family proteins were evaluated by Western blotting. Finally, to characterize entinostat's mechanisms-of-action, lymphoma cells were exposed to entinostat with or without pan-caspase (Q-VD-OPh, 5mM) and changes in cell viability were detected. Entinostat exhibited dose-dependent activity as a single agent against RSCL, RRCL and patient-derived primary tumor cells (N=32). In addition, in vitro exposure of lymphoma cells to entinostat resulted in an increase in G1 and a decrease in S phase. Moreover synergistic activity was observed by combining entinostat with BTZ in vitro. The pharmacological interactions between entinostat and proteasome inhibitor could be explained in part by each agent's effects on the expression levels of cell cycle proteins. In vitro exposure of lymphoma cells to entinostat resulted in p21 upregulation and p53 down-regulation, whereas BTZ exposure lead to up-regulation of Bak and Noxa and downregulation of Mcl-1 and Bcl-XL. Caspase inhibition diminished entinostat anti-tumor activity in RSCL but not in RRCL. Together this data suggests that entinostat has a dual mechanism-of-action and can induce cell death by caspase-dependent and independent pathways. Our data suggests that entinostat as a single agent is active against rituximab-chemotherapy sensitive and resistant lymphoma cells and potentiates the anti-tumor activity of BTZ. A better understanding in the molecular events (caspase-dependent and -independent) triggered by entinostat in combination with proteasome inhibition is important in order to develop optimal combination strategies using these novel agents in future clinical trials.

Disclosures:

Czuczman: Millennium: Honoraria, Research Funding. Hernandez-Ilizaliturri:Genmab: Research Funding; Amgen: Research Funding; Celgene: Consultancy.