Department of Experimental Hematopathology, Institute of Pathology, Charité Medical University, Berlin, Charitéplatz 1, 10117, Berlin, Germany.
Berlin School of Integrative Oncology, Charité - Medical University of Berlin, Berlin, Germany.
BMC Cancer. 2020 May 14;20(1):427. doi: 10.1186/s12885-020-06937-8.
BACKGROUND: Enhancer of zeste homolog 2 (EZH2) is considered an important driver of tumor development and progression by its histone modifying capabilities. Inhibition of EZH2 activity is thought to be a potent treatment option for eligible cancer patients with an aberrant EZH2 expression profile, thus the indirect EZH2 inhibitor 3-Deazaneplanocin A (DZNep) is currently under evaluation for its clinical utility. Although DZNep blocks proliferation and induces apoptosis in different tumor types including lymphomas, acquired resistance to DZNep may limit its clinical application. METHODS: To investigate possible mechanisms of acquired DZNep resistance in B-cell lymphomas, we generated a DZNep-resistant clone from a previously DZNep-sensitive B-cell lymphoma cell line by long-term treatment with increasing concentrations of DZNep (ranging from 200 to 2000 nM) and compared the molecular profiles of resistant and wild-type clones. This comparison was done using molecular techniques such as flow cytometry, copy number variation assay (OncoScan and TaqMan assays), fluorescence in situ hybridization, Western blot, immunohistochemistry and metabolomics analysis. RESULTS: Whole exome sequencing did not indicate the acquisition of biologically meaningful single nucleotide variants. Analysis of copy number alterations, however, demonstrated among other acquired imbalances an amplification (about 30 times) of the S-adenosyl-L-homocysteine hydrolase (AHCY) gene in the resistant clone. AHCY is a direct target of DZNep and is critically involved in the biological methylation process, where it catalyzes the reversible hydrolysis of S-adenosyl-L-homocysteine to L-homocysteine and adenosine. The amplification of the AHCY gene is paralleled by strong overexpression of AHCY at both the transcriptional and protein level, and persists upon culturing the resistant clone in a DZNep-free medium. CONCLUSIONS: This study reveals one possible molecular mechanism how B-cell lymphomas can acquire resistance to DZNep, and proposes AHCY as a potential biomarker for investigation during the administration of EZH2-targeted therapy with DZNep.
背景：增强子结合锌指蛋白 2（EZH2）通过其组蛋白修饰功能被认为是肿瘤发生和发展的重要驱动因素。抑制 EZH2 活性被认为是一种有效的治疗选择，适用于具有异常 EZH2 表达谱的合格癌症患者，因此间接 EZH2 抑制剂 3-去氮杂胞苷 A（DZNep）目前正在评估其临床应用。尽管 DZNep 可阻断不同肿瘤类型（包括淋巴瘤）的增殖并诱导细胞凋亡，但对 DZNep 的获得性耐药可能会限制其临床应用。
方法：为了研究 B 细胞淋巴瘤获得性 DZNep 耐药的可能机制，我们通过长期用递增浓度的 DZNep（范围为 200 至 2000 nM）处理先前对 DZNep 敏感的 B 细胞淋巴瘤细胞系，生成了一个 DZNep 耐药克隆，并比较了耐药和野生型克隆的分子谱。使用流式细胞术、拷贝数变异分析（OncoScan 和 TaqMan 分析）、荧光原位杂交、Western blot、免疫组织化学和代谢组学分析等分子技术进行了这一比较。
结果：全外显子测序并未显示获得有生物学意义的单核苷酸变异。然而，拷贝数改变的分析表明，除其他获得的不平衡外，耐药克隆中 S-腺苷-L-同型半胱氨酸水解酶（AHCY）基因的扩增（约 30 倍）。AHCY 是 DZNep 的直接靶标，并且在生物甲基化过程中起关键作用，其中它催化 S-腺苷-L-同型半胱氨酸可逆水解为 L-同型半胱氨酸和腺苷。AHCY 基因的扩增与转录和蛋白质水平的 AHCY 强烈过表达平行，并在耐药克隆在无 DZNep 的培养基中培养时仍然存在。
结论：本研究揭示了 B 细胞淋巴瘤获得对 DZNep 耐药的一种可能的分子机制，并提出 AHCY 作为在 DZNep 靶向 EZH2 治疗中进行研究的潜在生物标志物。
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