Combined Targeting of Chromatin Modifying Enzymes LSD1, EZH2 and Histone Deacetylases (HDACs) Has Superior Efficacy Against Human Mantle Cell Lymphoma Cells

Abstract

Abstract 2429PRC (polycomb repressive complex) 2 contains three core protein components, i.e., EZH2, SUZ12 and EED, of which EZH2 has the SET domain with its intrinsic histone methyltransferase activity that mediates the trimethylation (Me3) of lysine (K) 27 on histone (H) 3-a repressive chromatin mark for gene expression. We have previously reported that treatment with the S-adenosylhomocysteine hydrolase and EZH2 inhibitor, DZNep as well as treatment with the pan-histone deacetylase inhibitor panobinostat (PS, Novartis Pharma) deplete PRC2 complex proteins. LSD1 (KDM1A) is a demethylase of H3K4Me2, and inhibiton of LSD1 leads to increase in H3K4Me3-a permissive mark for gene expression. In the present studies, we determined the chromatin-modifying and cytotoxic effects of LSD1 inhibition alone and in combination with PS or DZNep in cultured (JeKo-1 and Z138C) and primary human Mantle Cell Lymphoma (MCL) cells. Treatment with the non-amine oxidase reversible inhibitor of LSD1 CIT0665 (250 to 1000 nM), or the more potent analogue HCI2509 (20 to 250 nM), dose-dependently increased the levels of H3K4Me2 & Me3, p21 and p27, while decreasing the levels of cyclin D1, which was associated with inhibition of cell proliferation and accumulation of the MCL cells in the G1 phase of the cell cycle. Abrogation of LSD1 by a specific shRNA treatment also induced similar chromatin, cell cycle and growth inhibitory effects. Exposure to CIT0665 or HCI2509 disrupted the binding of LSD1 with the co-repressor CoREST and HDAC1, without affecting the levels of these proteins. As noted above, treatment with PS (10 to 50 nM) dose-dependently depleted the levels of not only EZH2, SUZ12 and the PRC1 complex protein BMI1, but also of LSD1 in MCL cells. PS treatment alone also depleted the levels of AKT, cRAF, CDK4 and cyclin D1, as well as induced cell cycle growth inhibition and apoptosis of MCL cells. Co-treatment with PS enhanced the chromatin modifying effects of CIT0665 or HCI2509. The combination synergistically induced apoptosis of the cultured MCL cells (combination indices, CI <1.0). This was associated with greater induction of p27 and depletion of cyclin D1. Treatment with PS and HCI2509 also synergistically induced loss of viability of primary MCL cells (CI <1.0). We have previously reported that DZNep dose-dependently depleted EZH2, SUZ12 and BMI1 expression, inhibited H3K27Me3 levels, induced p21, p27 and FBXO32 (muscle atrophy F-box protein, also called atrogin-1) levels in cultured and primary MCL cells. Here, we determined the effects of co-treatment with HCI2509 and DZNep in MCL cells. Combined treatment with HCI2509, although not synergistic, enhanced the apoptosis of MCL cells induced by DZNep. Taken together these findings indicate that combined targeted depletion of the level and activity of LSD1 by PS and CIT0665 or HCI2509 along with PS-mediated depletion of PRC2 proteins, BMI and HDACs exerts superior activity against MCL cells. These studies also support the in vivo testing of combined epigenetic therapies in the treatment of MCL. Disclosures:Sharma:Novartis: Research Funding.