The Pan-Deacetylase Inhibitor Panobinostat Downregulates HIF-1α and VEGF and, Synergizes with Everolimus In Hodgkin Lymphoma Cell Lines

Abstract 2851

Deacetylases (DAC) inhibitors are promising new class of anticancer agents. Panobinostat (LBH589) is a pan-DAC inhibitor with evidence of clinical activity in patients with relapsed classical Hodgkin Lymphoma (HL). However the mechanisms of action of this new drug are not completely understood. The aim of this study was to investigate the mechanisms of antiproliferative effect induced by LBH589 in HL-derived cell lines. Experiments were performed in HL derived-cell lines (HDLM-2, KM-H2 and L-428). Cells were treated with 0.01–1μM of LBH589 alone or in combination with 0.01–1μM of everolimus (RAD001), an inhibitor of the mammalian target of rapamycin (mTOR), for 24–72 hours. Growth inhibition and apoptosis were analyzed in response to treatment using MTS assay, Western blotting and flow cytometric analyses. Effect of panobinostat on a panel of 30 cytokines and chemokines was assayed on cells after incubation of 24 hours using a multiplex assay. Panobinostat demonstrated antiproliferative activity in HL cell lines in a dose-and time-dependant manner with an IC50 ranging between 20 and 40nM at 72 hours. The antiproliferative activity was associated with downregulation of the X-linked inhibitor of apoptosis protein (XIAP), activation of caspase 9 and caspase 3, cleavage of Poly ADP Ribosome polymerase (PARP) and induction of apoptosis. In addition, panobinostat downregulated the level of the transcription factor hypoxia-inducible factor 1 alpha (HIF-1α) a major regulator of tumor cell adaptation to hypoxic stress. Furthermore, panobinostat decreased the level of the vascular endothelial growth factor (VEGF), an HIF-1α target gene, in the cell culture supernatants.The combination of panobinostat and everolimus had synergistic antiproliferative activity in the cells lines. This synergy was due to reciprocal inhibition of negative feedback loops induced by mTOR inhibitor and panobinostat therapy. Collectively, our data demonstrate that panobinostat induces apoptosis in HL cell lines by modulating several survival pathways. Furthermore, the observed synergy between panobinostat and everolimus provides rationale for combining these two active agents in a phase I/II clinical trial in patients with relapsed HL.