Vorinostat, a Histone Deacetylase (HDAC) Inhibitor, Is Active in Chemotherapy Resistant B-Cell Non-Hodgkin Lymphoma and Enhances the Anti-Tumor Activity of Chemotherapy Agents

Field: Pharmacology, Toxicology and Pharmaceutics (Life Sciences)

Abstract Pre-clinical models of chemotherapy resistance and clinical observations derived from the CORAL study suggest that primary refractory/relapsed B-Cell Non-Hodgkin Lymphoma (NHL) is a more aggressive and resistant lymphoma to current available treatments. Pre-clinically, we demonstrated that chemotherapy resistance is associated with a deregulation on the apoptotic machinery rendering lymphoma cells resistant to apoptotic stimuli. There is a dire need to develop agents capable to execute alternative pathways of cell death in an attempt to overcome chemotherapy resistance. Post-transcriptional histone modification plays an important role in regulating gene transcription, and is altered by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs regulate several key cellular functions, including: cell proliferation, cell cycle, apoptosis, angiogenesis, migration, antigen presentation, and/or immune regulation. Given their influence in multiple regulatory pathways, HDAC inhibition is an attractive strategy to evaluate its anti-tumor activity in cancer cells. To this end we studied the anti-tumor activity and mechanisms of action of suberoylanilide hydroxamic acid (SAHA, Vorinostat) in drug-resistant pre-clinical models. A panel of rituximab and chemotherapy resistant cell lines and primary tumor cells isolated from B-cell NHL patients (N=42: Denovo=27, Relapse/Rafractory=15) were exposed to escalating doses of vorinostat. Changes in cell viability were determined by Cell Titer Glo ® (Promega, Fitchburg, WI) luminescent assays. Changes in cell cycle were determined by flow cytometric analysis. Subsequently, protein lysates were isolated from vorinostat exposed cells and changes in members of apoptosis, cell cycle family proteins and the acetylation status of histone H3 were evaluated by Western blotting. In addition, cell lines were pre-exposed to vorinostat for 48 hrs and subsequently plated in 384 well plates and exposed to several chemotherapy agents (cisplatin, etoposide, or gemcitabine) changes in cell viability were determined by Cell Titer Glo and synergistic activity was evaluated using the Calcusm® software. Vorinostat induced dose-and time- dependent cell death in cell lines and in primary tumor cells. In addition, in vitro exposure of lymphoma cells to vorinostat resulted in an increase in p21, decrease cyclin dependent kinase2 (CDK2) and cyclin E; acetylation of histone H3 and was associated with a G1 cell cycle arrest. As expected, PARP cleavage was not observed in cell lines exposed to vorisnostat at IC50 doses, and Caspase inhibitor experiment further showed that blockage of caspase pathway cannot rescue cells and primary patient tumor samples from vorinostat induced cell death, suggesting that alternative cell death pathways were executed (i.e. irreversible cell cycle arrest). Moreover vorinostat was found to enhances the anti-tumor activity of chemotherapy agents. Our data suggests that vorinostat is active in drug-resistant pre-clinical models and that in cell lines with known defective apoptotic machinery, it can active alternative pathway of cell death. Given the multiple pathways affected by HDAC inhibition, vorisnostat can potentially be used to overcome acquired resistant to chemotherapy in aggressive B-cell lymphoma. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund) Disclosures No relevant conflicts of interest to declare.