Division of Hematology, Department of Internal Medicine, Mayo Clinic, College of Medicine, Rochester, MN, USA.
Department of Molecular and Experimental Therapeutics, Mayo Clinic, College of Medicine, Rochester, MN, USA.
Leukemia. 2014 Jan;28(1):147-54. doi: 10.1038/leu.2013.251. Epub 2013 Aug 27.
Protein tyrosine phosphatases such as PTPN6 can be downregulated in various neoplasms. PTPN6 expression by immunohistochemistry in 40 diffuse large B-cell lymphoma (DLBCL) tumors was lost or suppressed in 53% (21/40). To elucidate the molecular mechanisms of PTPN6 suppression, we performed a comprehensive epigenetic analysis of PTPN6 promoter 2 (P2). None of the DLBCL primary tumors (0/37) had PTPN6 hypermethylation on the CpG1 island using methylation-specific PCR, pyrosequencing, and high-resolution melting assays. However, hypermethylation in 57% (21/37) of cases was found in a novel CpG island (CpG2) in P2. PTPN6 gene suppression was reversed by 5-aza-deoxycytidine (5-Aza), a DNA methyltransferase inhibitor, and the histone deacetylase inhibitor (HDACi) LBH589. LBH589 and 5-Aza in combination inhibited DLBCL survival and PTPN6 hypermethylation at CpG2. The role of histone modifications was investigated with a chromatin-immunoprecipitation assay demonstrating that PTPN6 P2 is associated with silencing histone marks H3K27me3 and H3K9me3 in DLBCL cells but not normal B cells. 3-Deazaneplanocin A, a histone methyltransferase inhibitor, decreased the H3K27me3 mark, whereas HDACi LBH589 increased the H3K9Ac mark within P2 resulting in re-expression of PTPN6. These studies have uncovered novel epigenetic mechanisms of PTPN6 suppression and suggest that PTPN6 may be a potential target of epigenetic therapy in DLBCL.
蛋白酪氨酸磷酸酶（如 PTPN6）在各种肿瘤中可以下调。在 40 例弥漫性大 B 细胞淋巴瘤（DLBCL）肿瘤中，免疫组化检测到 PTPN6 的表达缺失或受抑占 53%（21/40）。为了阐明 PTPN6 抑制的分子机制，我们对 PTPN6 启动子 2（P2）进行了全面的表观遗传学分析。使用甲基化特异性 PCR、焦磷酸测序和高分辨率熔解分析，没有一个 DLBCL 原发性肿瘤（0/37）在 CpG1 岛上存在 PTPN6 高甲基化。然而，在 P2 的一个新的 CpG 岛（CpG2）中，57%（21/37）的病例存在高甲基化。DNA 甲基转移酶抑制剂 5-氮杂-2′-脱氧胞苷（5-Aza）和组蛋白去乙酰化酶抑制剂（HDACi）LBH589 可逆转 PTPN6 基因抑制。LBH589 和 5-Aza 联合抑制 DLBCL 存活和 CpG2 处的 PTPN6 高甲基化。通过染色质免疫沉淀实验研究了组蛋白修饰的作用，该实验表明 PTPN6 P2 与 DLBCL 细胞中的沉默组蛋白标记 H3K27me3 和 H3K9me3 相关，但与正常 B 细胞无关。组蛋白甲基转移酶抑制剂 3-去氮杂胞苷（3-Deazaneplanocin A）降低了 H3K27me3 标记，而 HDACi LBH589 增加了 P2 内的 H3K9Ac 标记，导致 PTPN6 重新表达。这些研究揭示了 PTPN6 抑制的新的表观遗传学机制，并表明 PTPN6 可能是 DLBCL 表观遗传治疗的潜在靶点。
