Efficacy of Combined Epigenetic Targeting of Histone Methyltransferase EZH2 and Histone deacetylases Against Human Mantle Cell Lymphoma Cells
Authors: Fiskus Warren , Rao Rekha , Balusu Ramesh , Tao Jianguo , Sotomayor Eduardo M. , Atadja Peter , Bhalla Kapil N. 
Published: October 2019

Abstract
Abstract 2488
Lysine specific histone methylation and deacetylation are chromatin modifications that, along with DNA methylation, are involved in the epigenetic silencing of tumor suppressor genes (TSGs). This silencing is mediated by multi-protein complexes PRC (polycomb repressive complexes) 1 and 2. Of the three core protein components of PRC2, i.e., EZH2, SUZ12 and EED, EZH2 has the SET domain with its intrinsic histone methyltransferase activity, which induces the trimethylation (Me3) of lysine (K) 27 on histone (H) 3-a repressive histone modification mediating gene repression. The PRC1 components include BMI1, MEL18, RING1 and RING2, and it serves to further compact the chromatin at PRC2 target genes. The RING1 and RING2 proteins are responsible for the ubiquitylation of K119 on H2A. We have previously reported that treatment with the pan-histone deacetylase inhibitor panobinostat (PS, Novartis Pharmaceutical Corp) depletes PRC2 complex proteins EZH2 and SUZ12 and the DNA methyltransferase (DNMT) 1. We also showed that co-treatment with the S-adenosylhomocysteine hydrolase and EZH2 inhibitor, DZNep, further depleted PRC2 complex proteins and, in combination with PS, induced synergistic apoptosis of cultured and primary AML cells (Blood 2009; 114: 2733–43). In the present studies we determined the effects of DZNep and/or PS on the expression of PRC1 and PRC2 proteins in human Mantle Cell Lymphoma (MCL) cells. Treatment with DZNep dose-dependently depleted EZH2, SUZ12 and BMI1 expression as well as inhibited K27Me3, while inducing K27 acetylation on H3. DZNep treatment also induced p21, p27 and FBXO32, while depleting the levels of cyclin D1 in the cultured MCL JeKo-1 and MO2058 cells. Similar induction of p21, p27 and FBXO32 were also observed, following siRNA knockdown of EZH2 in the cultured MCL cells. Notably, DZNep also induced similar perturbations in primary, patient-derived MCL cells. Treatment with PS alone attenuated EZH2, SUZ12 and DNMT1, as well as depleted the protein expression of BMI1, RING2 and MEL18 in the cultured MCL cells. This was associated with attenuation of H3K27Me3 and augmentation of H3K4Me3 chromatin marks. PS treatment also induced heat shock protein (hsp) 90 acetylation, and depleted the levels of hsp90 client proteins in the MCL cells, including CDK4, c-RAF and AKT. As compared to treatment with each agent alone, co-treatment with DZNep and PS caused greater depletion of EZH2, SUZ12 and BMI1, accompanied with greater induction of p21 and p27 but attenuation of cyclin D1 expression. Co-treatment with DZNep and PS also induced cell cycle growth arrest and synergistically induced apoptosis of JeKo-1 and MO2058, as well as of primary MCL cells derived from 3 patients with MCL (combination indices <1.0). Taken together these findings indicate that by targeted depletion of the PRC2 and PRC1 components and associated chromatin and other protein modifications (hsp90 acetylation), co-treatment with DZNep and PS represents a superior therapy of human MCL cells. These studies also support the in vivo testing of combined epigenetic therapies involving agents that target deregulated epigenetic mechanisms, e.g., histone deacetylases, methyl transferases and demethylases, as well as target DNMTs in the therapy of MCL.

Disclosures:
Atadja: Novartis: Employment.