Expression data from EZH2 inhibitor treated Non-Hodgkins Lymphoma cell lines

Mutations within the catalytic domain of the histone methyltransferase (HMT) EZH2 have been identified in subsets of Non-Hodgkin Lymphoma (NHL) patients. These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in these cancers. We previously reported the discovery of a potent, selective, S-adenosyl-methionine-competitive and orally bioavailable small molecule inhibitor of EZH2, EPZ-6438. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) led to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of xenograft-bearing mice with EPZ-6438 leads to dose-dependent tumor growth inhibition and eradication of genetically altered NHL with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 day after stopping compound treatment in two EZH2 mutant xenograft models. These data confirm the dependency of mutant NHL on EZH2 activity and portend the utility of EZH2-targeted drugs for the treatment of these genetically defined cancers. To identify potential biomarker and gain mechanistic insights in EPZ6438 treated lymphoma, lymphoma cell lines with or without EZH2 Y641 mutation were treated with EPZ6438 at Lowest Cytotoxic Concentration (LCC) and 10xLCC. Transcriptomes were profiled on Affemetrix U133 Plus2 chips. Differentially expressed genes and pathways upon compound treatment were anayzed. Lymphoma cell lines WSU-DLCL2, SU-DHL6, Pfeiffer, and Karpas422 were treated by EPZ6438 for 2 days, 4 days and 6 days. Treatment doses are LCC and 10xLCC of each cell lines. KARPAS_422, SUDHL6, and WSU_DLCL2 have the EZH2 Y641 mutation. PFEIFFER does not have the EZH2 Y641 mutation.

组蛋白甲基转移酶（histone methyltransferase, HMT）EZH2的催化结构域内的突变，已在非霍奇金淋巴瘤（Non-Hodgkin Lymphoma, NHL）患者的多个亚群中被鉴定出。此类遗传变异被推测可使这类癌症对EZH2的酶活性产生致癌性依赖。我们此前曾报道过一种强效、选择性、S-腺苷甲硫氨酸（S-adenosyl-methionine）竞争性且口服生物可利用的EZH2小分子抑制剂EPZ-6438的发现。在EZH2野生型与突变型淋巴瘤细胞中，EPZ-6438均可通过浓度与时间依赖的方式，选择性抑制细胞内组蛋白H3赖氨酸27（H3K27）的甲基化修饰。组蛋白H3赖氨酸27三甲基化（H3K27Me3）的抑制作用，可选择性杀伤携带EZH2催化结构域点突变的人淋巴瘤细胞系。向携带异种移植瘤的小鼠施用EPZ-6438，可实现剂量依赖性的肿瘤生长抑制，并根除携带遗传变异的NHL，同时伴随肿瘤及选定正常组织中H3K27Me3水平的相应降低。在两种EZH2突变型异种移植瘤模型中，经口给予EPZ-6438连续28天的小鼠，在停止给药后最长63天仍未出现肿瘤。上述数据证实了突变型NHL对EZH2活性的依赖性，并预示了靶向EZH2的药物在治疗这类基因特征明确的癌症中的应用潜力。为识别EPZ6438治疗淋巴瘤的潜在生物标志物并获取机制研究见解，我们将携带或不携带EZH2 Y641突变的淋巴瘤细胞系，分别以最低细胞毒浓度（Lowest Cytotoxic Concentration, LCC）及其10倍浓度（10xLCC）进行EPZ-6438处理。利用Affymetrix U133 Plus2芯片对转录组进行了分析。对化合物处理后的差异表达基因及通路进行了分析。淋巴瘤细胞系WSU-DLCL2、SU-DHL6、Pfeiffer及Karpas422分别经EPZ-6438处理2天、4天及6天，给药剂量为各细胞系对应的LCC与10xLCC。其中KARPAS_422、SUDHL6及WSU_DLCL2携带EZH2 Y641突变，而PFEIFFER不携带该突变。