Abstract 2016: Combined targeting of epigenetic mechanisms has superior efficacy against human mantle cell lymphoma cells
Authors: Fiskus Warren C. , Sharma Sunil , Rao Rekha , Balusu Ramesh , Tao Jianguo , Sotomayor Eduardo , Atadja Peter , Ganguly Siddhartha , Bearss David , Bhalla Kapil N. 
Published: July 2012

Abstract
Gene 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. This induces the trimethylation (Me3) of lysine (K) 27 on histone (H) 3-a repressive chromatin mark for gene repression. The PRC1 components include BMI1, RING1 and RING2, which are responsible for the ubiquitylation (Ub) of K119 on H2A and compaction of the chromatin at PRC2 target genes. We have previously reported that treatment with the pan-histone deacetylase inhibitor panobinostat (PS, Novartis Pharma) depletes EZH2, 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. In the present studies we determined that DZNep dose-dependently depleted EZH2, SUZ12 and BMI1 expression as well as inhibited K27Me3 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. 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 H2K119Ub, and augmentation of H3K4Me3 chromatin marks. Depletion of BMI with shRNA also reduced the levels of H2K119 Ub, which was associated with growth inhibition of MCL cells. PS treatment also decreased the levels of LSD1, a demethylase of H3K4Me2, which led to increase in H3K4Me3. Treatment with the LSD1 inhibitor CIT0665, or knockdown of LSD1 by shRNA, increased H3K4Me2 &Me3 levels, as well as enhanced PS mediated growth inhibition and apoptosis of MCL cells. PS treatment also induced heat shock protein (hsp) 90 acetylation, and depleted the levels of hsp90 client proteins in JeKo-1, 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, MO2058, and primary MCL cells derived from 3 patients with MCL (combination indices &lt;1.0). Taken together these findings indicate that combined targeted depletion of the levels and activities of EZH2, HDACs, LSD1 and BM1 exerts superior activity against MCL cells. These studies also support the in vivo testing of combined epigenetic therapies in the therapy of MCL.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2016. doi:10.1158/1538-7445.AM2011-2016