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Full Text (PDF) Published OnlineFirst August 29, 2012; DOI: 10.1158/1078-0432.CCR-12-0873 Clinical Cancer Cancer Therapy: Preclinical Research Superior Efficacy of a Combined Epigenetic Therapy against Human Mantle Cell Lymphoma Cells Warren Fiskus1, Rekha Rao1, Ramesh Balusu1, Siddhartha Ganguly1, Jianguo Tao2, Eduardo Sotomayor2, Uma Mudunuru1, Jacqueline E. Smith1, Stacey L. Hembruff1, Peter Atadja3, Victor E. Marquez4, and Kapil Bhalla1 Abstract Purpose: A deregulated epigenome contributes to the transformed phenotype of mantle cell lymphoma (MCL). This involves activity of the polycomb repressive complex (PRC) 2, containing three core proteins, EZH2, SUZ12, and EED, in which the SET domain of EZH2 mediates the histone methyltransferase activity. We determined the effects of 3-deazaneplanocin A (DZNep), an S-adeno- sylhomocysteine hydrolase inhibitor, and/or pan-histone deacetylase inhibitor panobinostat (PS) on cultured and primary MCL cells. Experimental Design: Following treatment with DZNep and/or PS, apoptosis and the levels and activity of EZH2 and PRC2 proteins in cultured and primary MCL cells were determined. Results: Treatment with DZNep depleted EZH2, SUZ12, and 3MeK27H3 in the cultured human MCL cells. DZNep also increased expression of p21, p27, and FBXO32, whereas it depleted Cyclin D1 and Cyclin E1 levels in MCL cells. In addition, DZNep treatment induced cell-cycle arrest and apoptosis in cultured and primary MCL cells. Furthermore, as compared with treatment with each agent alone, cotreatment with DZNep and PS caused greater depletion of EZH2, SUZ12, 3MeK27H3, and Cyclin D1 levels, whereas it induced greater expression of FBXO32, p16, p21, and p27. Combined treatment with DZNep and PS synergistically induced apoptosis of cultured and primary MCL cells while relatively sparing normal CD34 þ cells. Cotreatment with DZNep and PS also caused significantly greater inhibition of tumor growth of JeKo-1 xenografts in NOD/SCID mice. Conclusions: These preclinical in vitro and in vivo findings show that cotreatment with DZNep and PS is an active combined epigenetic therapy worthy of further in vivo testing against MCL. Clin Cancer Res; 18(22); 6227–38. Ó2012 AACR. Introduction the pathogenesis of MCL (3–5). In addition, increased Mantle cell lymphoma (MCL) is an aggressive and dis- expression of MYC, Cyclin D1, and EZH2 has been associ- tinct type of B-cell malignancy that comprises up to 10% of ated with poor prognosis and correlate with poorer clinical non-Hodgkin lymphomas (1). A characteristic feature of outcome in MCL (3, 6–8). EZH2 is a core member and the MCL is the chromosomal rearrangement caused by the catalytic subunit of the polycomb protein complex, poly- translocation t(11;14)(q13;q32), which results in overex- comb repressive complex (PRC) 2, that also contains SUZ12 pression of Cyclin D1 (2, 3). Copy number variations and and EED, in which the SET domain of EZH2 mediates the associated gene expression changes involving CDKN2A histone lysine (K) methyltransferase (KMTase) activity of (p16) and CDKN2B (p15), as well as altered expression of PRC2 (9). EZH2 is a KMTase that mediates epigenetic CDK4, and p27 has also been noted and may play a role in silencing of PRC2 target genes by inducing trimethylation (3Me) of K27 on histone H3 (3MeK27H3) in the chromatin (9, 10). EZH2 overexpression in transformed cells promotes Authors' Affiliations: 1University of Kansas Cancer Center, Kansas City, cell proliferation and aggressiveness (11–13). In hemato- Kansas; 2H. Lee Moffitt Cancer Center, Tampa, Florida; 3Novartis Institute HOX for Biomedical Research, Inc., Cambridge, Massachusetts; and 4National logic malignancies, PRC2 regulates the expression of Institutes of Health, Bethesda, Maryland genes and epigenetically represses genes including tumor Note: Supplementary data for this article are available at Clinical Cancer suppressor genes p16 (CDKN2A) and p14 (ARF; ref. 14). Research Online (http://clincancerres.aacrjournals.org/). EZH2 was shown to be preferentially overexpressed in Corresponding Author: Kapil Bhalla, University of Kansas Cancer Center, proliferating but not resting MCL cells (15). Recently, in 3901 Rainbow Blvd., 4030 Robinson, MS 1027, Kansas City, KS 66160. lymphomagenesis, EZH2-mediated gene silencing in ger- Phone: 913-945-7086; Fax: 913-588-4701; E-mail: [email protected] minal center B cells was shown to contribute to cell prolif- doi: 10.1158/1078-0432.CCR-12-0873 eration (16). Somatic gain of function mutations in tyrosine Ó2012 American Association for Cancer Research. 641 (Y641C) in EZH2 have also been noted to selectively www.aacrjournals.org 6227 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst August 29, 2012; DOI: 10.1158/1078-0432.CCR-12-0873 Fiskus et al. mined in MCL cells. In contrast, treatment with hydroxa- Translational Relevance mate pan-histone deacetylase (HDAC) inhibitors, includ- Overexpression of EZH2 and the polycomb repressive ing vorinostat and panobinostat (PS, LBH589, Novartis complex (PRC) 2 proteins has been associated with poor Pharmaceutical Inc.), has been shown to be active against prognosis and correlates with poorer clinical outcome in MCL cells (29–31). In acute myelogenous leukemia (AML) mantle cell lymphoma (MCL). PRC2 mediates trimethy- cells, treatment with PS was shown to deplete the levels of lation of lysine 27 on histone H3 (3MeK27H3) and EZH2 and SUZ12, as well as reduce the 3MeK27H3 mark on epigenetically represses genes such as the tumor sup- the chromatin (32). PS treatment also depleted the levels of pressor p16. The effects of pharmacologic inhibition of DNMT1 and disrupted its binding to EZH2 in AML cells (33, EZH2 or EZH2 knockdown have not been tested in MCL 34). In addition, cotreatment with DZNep and PS was cells. Here, we show that treatment with the EZH2 shown to be more effective against the epigenetic targets antagonist DZNep or the pan-histone deacetylase inhib- and synergistically induced apoptosis of AML cells (34). In itor panobinostat (PS) depletes EZH2, SUZ12, and the present studies, we determined that treatment with 3MeK27H3. Cotreatment with PS-augmented DZNep DZNep depletes PRC2 complex proteins and induces mediated attenuation of PRC2, which was associated cell-cycle arrest and apoptosis of cultured and primary MCL with synergistic activity of the combination against cells. Our findings also show that the combined treatment human MCL cells in vitro and in vivo. These observations of DZNep and PS causes more depletion of PRC2 complex support the rationale to further test the in vivo anti-MCL proteins and synergistically induces apoptosis of cultured efficacy of the combination of PS with an EZH2 antag- and primary MCL cells but not normal CD34 þ bone onist or combination therapies that target the deregu- marrow progenitor cells. In addition, cotreatment with lated epigenome in human MCL cells. DZNep and PS was more effective than each agent alone in inhibiting the in vivo tumor growth of JeKo-1 xenografts in NOD/SCID mice. alter PRC2 catalytic activity and drive hypertrimethylation Materials and Methods of H3K27 in germinal center B cell lymphoma, thereby Reagents identifying EZH2 as a potentially attractive therapeutic tar- PS was kindly provided by Novartis Pharmaceuticals, Inc. get (17–20). PRC2-mediated trimethylation of H3K27 also DZNep was acquired from the National Cancer Institute recruits the multiprotein PRC1 complex, consisting among (Rockville, MD). Monoclonal BMI1, polyclonal trimethy- others of B lymphoma Mo-MLV insertion region 1 homolog lated K9 histone H3, polyclonal trimethylated K79 histone (BMI1), as well as RING1 and RING2 proteins, which H3, acetylated K16 histone H4, acetyl K56 histone H3, and mediate ubiquitylation of histone H2A on K119 associated polyclonal trimethylated K4 histone H3 antibodies were with gene repression (9, 14). BMI1 represses CDKN2A and purchased from Millipore. All other validated antibodies INK4A/ARF and cooperates with MYC in lymphomagenesis were purchased from vendors, as previously described (29, (14). In addition, the BMI1 locus has been shown to be 30, 33, 34). amplified in MCL cells (21). EZH2 has also been reported to directly control DNA methylation through its association Cell lines and cell culture with and regulation of the activity of the DNA methyltrans- MCL cell line MO2058 was obtained and maintained as ferases DNMT1, DNMT3a, and DNMT3b (22, 23). Howev- previously described (29, 30). JeKo-1 and Z-138 cells were er, genes silenced in cancer by 3MeK27H3 have been shown obtained from American Type Culture Collection (ATCC, to be independent of promoter DNA methylation indicating Manassas, VA). Both cell lines were banked after receipt, and that PRC2-mediated silencing could be an independent passaged for less than 6 months before use in these studies. mechanism for suppression of tumor suppressor genes The ATCC characterizes cell lines using short tandem repeat (24). Consistent with this, both DNA methylation and polymorphism analysis. MCL cells were maintained in transcriptional silencing of PRC2 target genes persists when culture, as previously described (29, 30). Logarithmically EZH2 expression is depleted (25, 26). growing cells were exposed to the designated concentrations 3-deazaneplanocin A (DZNep) is the cyclopentanyl ana- of DZNep and/or PS. Following these treatments, cells were log of 3-deazaadenosine that inhibits the activity of S- washed free of the drug(s) before the performance
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