Entinostat, a Novel Histone Deacetylase (HiDAC) Inhibitor Is Active in B-Cell Lymphoma and Enhances the Anti-Tumor Activity of Rituximab, Chemotherapy Agents and Proteasome Inhibitors
Authors: Frys Sarah , Barth Matthew J. , Mavis Cory , Gu Juan , Gibbs John , Czuczman Myron S. , Hernandez-Ilizaliturri Francisco 
Published: October 2019

Abstract 1649

Deacetylases (DACs) are enzymes that remove the acetyl groups from target protein lysines leading to regulation of gene transcription and other cellular processes. Entinostat is a novel and potent class I DAC inhibitor undergoing pre-clinical and clinical testing. We previously demonstrated that entinostat was active against a panel of rituximab-sensitive (RSCL) and –resistant cell lines (RRCL); and enhanced the anti-tumor activity of chemotherapy agents. In the current work, we studied the mechanisms-of-action of entinostat and its effect on rituximab activity and proteasome inhibition. Our results indicate that entinostat potentiates both rituximab and proteasome inhibitor activity in NHL cells through a dual caspase dependent and independent mechanism-of-action. Studies were conducted in RSCL, RRCL and in primary tumor cells isolated from patients with B-cell non-Hodgkin's lymphoma (n = 43). For studies with a proteasome inhibitor, cells were exposed to escalating doses of entinostat +/− bortezomib of up to 72 hrs. Studies were repeated with or without a pan-caspase inhibitor (Q-VD-OPh, 5uM). Changes in mitochondrial potential and ATP synthesis were determined by alamar blue reduction and cell titer glo luminescent assays, respectively. Flow cytometric analysis was used to determine changes in cell cycle. Protein lysates from entinostat +/− BTZ exposed cells were evaluated for changes in members of Bcl-2 and cell cycle family proteins. Microarray analysis was performed to discern differences in gene expression between RRCL and RSCL after incubation with entinostat. Separately, we studied the effects of entinostat in vitro and in vivo on rituximab-associated complement-mediated cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC). NHL cells were exposed to entinostat or DMSO 0.01% for 48hrs and then labeled with 51Cr. Labeled target cells were then exposed to rituximab or isotype control (10mg/ml) and human serum (25%) (CMC) or PBMCs (Effector:Target ratio, 40:1) (ADCC). For in vivo studies, 6–8 week old SCID mice were inoculated with 1′106 Raji cells via tail vein injection and after a period of 72 hours animals were divided into six cohorts: control, entinostat alone at 5mg/kg/dose, entinostat alone at 20mg/kg/dose alone, rituximab single agent at 10mg/kg/dose, rituximab and entinostat at 5mg/kg/dose and rituximab + entinostat at 20mg/kg/dose. Entinostat was administered by gastric lavage daily on days +3–8 and +11–16, and rituximab was administered via tail vein injection on days +3, +8, +11, and +16. Difference in survival between treatment groups was performed by Kaplan-Meier analysis. Entinostat exhibited dose-dependent activity as a single agent against RSCL, RRCL and patient-derived primary lymphoma cells. In vitro exposure of lymphoma cells to entinostat resulted in an increase in G1 and a decrease in S phase. Synergistic activity was observed by combining entinostat with BTZ but not rituximab in vitro. Microarray data demonstrated that p21 was up-regulated after exposure to entinostat. Additionally, we found an upregulation of ICAM2, down-regulation of E2F2 but no changes in CD20 mRNA. Findings were confirmed at the protein level. Caspase inhibition diminished entinostat anti-tumor activity in RSCL but not in RRCL suggesting that entinostat has a dual mechanism-of-action and can induce cell death by caspase-dependent and independent pathways (i.e. cell cycle arrest). Furthermore, In vivo treatment of SCID mice with high dose entinostat in combination with rituximab led to prolongation of the mean survival (87 days, 72–103 95% C.I. P=0.05) compared to rituximab (67 days, 52–82 95% C.I.) entinostat 20 mg/kg (19 days, 19–20 95% C.I.) alone. Our data suggests that entinostat is active against RSCL and RRCL and potentiates the anti-tumor activity of BTZ or rituximab. In addition, entinostat appears to have a dual mechanism of action that supports its clinical development in rituximab-sensitive or resistant lymphomas. Ongoing studies are aimed to further define the molecular events responsible for entinostat mechanisms-of-action and optimize its spectrum of anti-tumor activity. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute).

Disclosures:

No relevant conflicts of interest to declare.