RNF38 通过调节 LMX1A 的泛素化诱导自噬促进急性髓系白血病对吉特替尼的耐药性。
RNF38 promotes gilteritinib resistance in acute myeloid leukemia via inducing autophagy by regulating ubiquitination of LMX1A.
机构信息
Department of Oncology, Ganzhou Cancer Hospital, Gannan Medical University, Ganzhou City, 341000, Jiangxi Province, P.R. China.
Suzhou Medical College of Soochow University, Suzhou City, 215123, Jiangsu Province, P.R. China.
出版信息
Cell Biol Toxicol. 2024 Nov 28;40(1):105. doi: 10.1007/s10565-024-09936-8.
BACKGROUND
Gilteritinib is a commonly used targeted drug for acute myeloid leukemia (AML), but the emergence of gilteritinib resistance greatly reduces the therapeutic effect. RING finger protein 38 (RNF38), a protein with RING Finger domain and E3 ubiquitin ligase activity, has been implicated in tumorigenesis and drug resistance. However, the role and mechanism of RNF38 in the gilteritinib resistance of AML remains unclear.
METHODS
Normal AML cells were treated with gilteritinib to construct gilteritinib-resistant cells (MV4-11/Gilteritinib and MOLM-13/Gilteritinib). CCK8 assay, TUNEL staining and EdU assay were used to assess gilteritinib resistance, cell apoptosis and proliferation. The protein levels of autophagy-related markers, RNF38 and LIM homeobox transcription factor 1 alpha (LMX1A) were determined by western blot. Also, RNF38 and LMX1A mRNA levels were tested using qRT-PCR. Autophagic flux was assessed using mRFP-GFP-LC3 labeling, and autophagosome numbers was counted under transmission electron microscopy. Co-IP assay was employed to analyze interaction between RNF38 and LMX1A. The effects of LMX1A and RNF38 on AML tumorigenesis were analyzed by in vivo experiments.
RESULTS
In gilteritinib-resistant AML cells, autophagy-related markers, mRFP-GFP-LC3 signals and autophagosome numbers were significantly enhanced. Autophagy inhibitor 3-MA could suppress gilteritinib resistance in AML cells. RNF38 knockdown inhibited gilteritinib resistance and autophagy in AML cells. Mechanistically, RNF38 reduced LMX1A expression by inducing its ubiquitination. RNF38 overexpression reversed the inhibitory effect of LMX1A on gilteritinib resistance and autophagy in AML cells, as well as AML tumor growth in vivo, while these effects could be abolished by proteasome inhibitor MG132.
CONCLUSIONS
RNF38 induced autophagy to promote gilteritinib resistance in AML by increasing the ubiquitination of LMX1A.
背景
吉特替尼是一种常用于治疗急性髓系白血病（AML）的靶向药物，但吉特替尼耐药的出现大大降低了治疗效果。RING 指蛋白 38（RNF38）是一种具有 RING 指结构域和 E3 泛素连接酶活性的蛋白质，与肿瘤发生和耐药性有关。然而，RNF38 在 AML 中对吉特替尼耐药的作用和机制尚不清楚。
方法
用吉特替尼处理正常 AML 细胞，构建吉特替尼耐药细胞（MV4-11/吉特替尼和 MOLM-13/吉特替尼）。CCK8 测定、TUNEL 染色和 EdU 测定用于评估吉特替尼耐药、细胞凋亡和增殖。Western blot 测定自噬相关标记物、RNF38 和 LIM 同源框转录因子 1 阿尔法（LMX1A）的蛋白水平。还使用 qRT-PCR 测试 RNF38 和 LMX1A mRNA 水平。使用 mRFP-GFP-LC3 标记评估自噬流，并在透射电子显微镜下计数自噬体数量。采用 Co-IP 测定分析 RNF38 与 LMX1A 之间的相互作用。体内实验分析 LMX1A 和 RNF38 对 AML 肿瘤发生的影响。
结果
在吉特替尼耐药的 AML 细胞中，自噬相关标记物、mRFP-GFP-LC3 信号和自噬体数量显著增强。自噬抑制剂 3-MA 可抑制 AML 细胞中的吉特替尼耐药。RNF38 敲低抑制 AML 细胞中的吉特替尼耐药和自噬。机制上，RNF38 通过诱导其泛素化降低 LMX1A 的表达。RNF38 过表达逆转了 LMX1A 对 AML 细胞中吉特替尼耐药和自噬的抑制作用，以及体内 AML 肿瘤生长，而这些作用可被蛋白酶体抑制剂 MG132 所消除。
结论
RNF38 通过增加 LMX1A 的泛素化诱导自噬来促进 AML 中的吉特替尼耐药。
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