Combined Epigenetic Therapy with the Novel Histone Methyl Transferase EZH2 Inhibitor 3-Deazaneplanocin and Histone Deacetylase Inhibitor Panobinostat Exerts Synergistic Activity against Human Mantle Cell Lymphoma Cells

Lysine specific histone methylation and deacetylation and DNA hypermethylation are involved in the epigenetic silencing of tumor suppressor genes (TSG), e.g., p16 and JunB. The multi-protein complex PRC (polycomb repressive complex) 2 that contains the three core proteins EZH2, SUZ12 and EED, has intrinsic histone methyltransferase (HMTase) activity. This is mediated by the SET domain of EZH2, which induces tri-methylation (3Me) of lysine (K)-27 on histone H3, as well as promotes cell proliferation and aggressiveness of neoplastic cells. EZH2 is preferentially overexpressed in proliferating but not resting Mantle Cell Lymphoma (MCL) cells. In the present studies we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) dose-dependently (500 nM to 2.0 uM) depletes EZH2, SUZ12 and EED levels, as well as inhibits 3Me K27 on H3 while inducing K27 H3 acetylation. DZNep treatment also induces the levels of p21, p27, JunB and FBXO32, while depleting cyclin D1 and cyclin E levels in the cultured human MCL Jeko-1, MO2058 and Z138 cells and in primary patient-derived MCL cells. Treatment with DZNep induces PARP cleavage activity of the caspases and apoptosis in the cultured and primary MCL cells. DZNep promoted proteasomal degradation of EZH2 and SUZ12, since co-treatment with bortezpmib significantly restored EZH2 and SUZ12 levels in the MCL cells. We had previously reported that treatment with the pan-histone deacetylase (HDAC) inhibitor panobinostat (PS) (LBH589, Novartis Pharmaceutical Corp) depletes the levels of EZH2, SUZ12 and EED in cultured and primary AML cells (Mol Cancer Ther. 2006; 5:3096). Within the PRC2 complex, EZH2 bound and recruited the DNA methyltransferases DNMT1, and treatment with PS also disrupted the interaction of EZH2 with DNMT1, attenuated DNMT1 levels and its binding to the EZH2-targeted gene promoters, e,g, JunB. Here, we also demonstrate that, PS treatment depletes DNMT1 levels and induces JunB levels in cultured MCL cells. As compared to treatment with either agent alone, co-treatment with DZNep and PS caused more depletion of EZH2 and SUZ12, but not of DNMT1, more induction of JunB, p21 and p27, as well as synergistically induced apoptosis of cultured MCL cells (combination indices < 1.0). Taken together, these findings indicate that DZNep and PS mediated targeting of EZH2 and the PRC2 complex is an effective epigenetic therapy of MCL, which also results in undermining several molecular determinants of MCL cell proliferation and survival. Additionally, combined epigenetic therapy with DZNep and PS exerts synergistic in vitro activity against human MCL cells, suggesting that this combination may be a promising novel treatment for MCL.