Novel impact of the DNMT3A R882H mutation on GSH metabolism in a K562 cell model established by TALENs.
Novel impact of the DNMT3A R882H mutation on GSH metabolism in a K562 cell model established by TALENs.
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DOI：
10.18632/oncotarget.16449
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发表时间：
2017-05-02
期刊：
影响因子：
--
通讯作者：
Xiao M
中科院分区：
文献类型：
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作者：
Yang L;Liu Y;Zhang N;Ding X;Zhang W;Shen K;Huang L;Zhou J;Cui S;Zhu Z;Hu Z;Xiao M
DNA甲基转移酶3A（DNMT3A）突变发生在18%~23%的急性髓系白血病（AML）患者中，被认为是成人新发AML病例的不良预后因素。然而，AML发病机制中突变的相关分子机制仍不清楚。在本研究中，我们通过类转录激活因子效应核酸酶（TALEN）和成簇规则间隔短回文重复序列（CRISPR/Cas9）技术建立了携带DNMT3A R882H突变的K562和SKM1细胞模型，并发现突变的DNMT3A可以促进恶性细胞克隆的增殖能力。进一步的RNA微阵列分析显示，一些对谷胱甘肽（GSH）合成至关重要的基因，包括CTH、PSPH、PSAT1，尤其是SLC7A11（半胱氨酸/谷氨酸转运蛋白）显着上调，从而导致细胞内GSH水平显着升高。随后的实验表明，突变克隆对化疗和 SLC7A11 抑制剂具有抗性。通过 shRNA 诱导 SLC7A11 沉默，我们发现 DNMT3A 突变克隆的细胞 GSH 和细胞增殖能力显着降低。我们的研究结果为 DNMT3A R882H 突变在 AML 发病机制中的作用提供了新的见解，并表明针对细胞 GSH 合成途径可以增强目前对 DNMT3A R882H 突变的 AML 患者的治疗。
DNA methyltransferase 3A (DNMT3A) mutations occurred in 18%~23% of acute myeloid leukemia (AML) patients, and were considered to be an adverse prognostic factor for adult de novo AML cases. However, the relevant molecular mechanism of the mutation in AML pathogenesis remains obscure. In this study, we established K562 and SKM1 cell model carrying the DNMT3A R882H mutation via transcription activator-like effector nuclease (TALEN) and Clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) technology, and discovered that mutated DNMT3A could promote the proliferative capability of malignant cell clones. Further RNA microarray analysis revealed that some genes crucial for glutathione (GSH) synthesis, including CTH, PSPH, PSAT1 and especially SLC7A11 (the cysteine/glutamate transporter) were significantly up-regulated, which resulted in significant elevation of intracellular GSH levels. A subsequent experiment demonstrated that the mutant clones are resistant to chemotherapy as well as SLC7A11-inhibitorsBy shRNA induced SLC7A11 silencing, we discovered profoundly decreased cellular GSH and cell proliferative ability of DNMT3A mutated clones. Our results provided novel insight into the role of the DNMT3A R882H mutation in AML pathogenesis and suggested that targeting the cellular GSH synthetic pathway could enhance the current therapy for AML patients with the DNMT3A R882H mutation.