PDF(Pubmed)
Abstract：
BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic malignancy with poor outcomes, especially in older AML patients. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered a promising anticancer drug because it selectively induces the extrinsic apoptosis of tumor cells without affecting normal cells. However, clinical trials have shown that the responses of patients to TRAIL are significantly heterogeneous. It is necessary to explore predictable biomarkers for the preselection of AML patients with better responsiveness to TRAIL. Here, we investigated the critical role of tumor protein p53 inducible nuclear protein 2 (TP53INP2) in the AML cell response to TRAIL treatment.
METHODS: First, the relationship between TP53INP2 and the sensitivity of AML cells to TRAIL was determined by bioinformatics analysis of Cancer Cell Line Encyclopedia datasets, Cell Counting Kit-8 assays, flow cytometry (FCM) and cell line-derived xenograft (CDX) mouse models. Second, the mechanisms by which TP53INP2 participates in the response to TRAIL were analyzed by Western blot, ubiquitination, coimmunoprecipitation and immunofluorescence assays. Finally, the effect of TRAIL alone or in combination with the BCL-2 inhibitor venetoclax (VEN) on cell survival was explored using colony formation and FCM assays, and the effect on leukemogenesis was further investigated in a patient-derived xenograft (PDX) mouse model.
RESULTS: AML cells with high TP53INP2 expression were more sensitive to TRAIL in vitro and in vivo. Gain- and loss-of-function studies demonstrated that TP53INP2 significantly enhanced TRAIL-induced apoptosis, especially in AML cells with nucleophosmin 1 (NPM1) mutations. Mechanistically, cytoplasmic TP53INP2 maintained by mutant NPM1 functions as a scaffold bridging the ubiquitin ligase TRAF6 to caspase-8 (CASP 8), thereby promoting the ubiquitination and activation of the CASP 8 pathway. More importantly, simultaneously stimulating extrinsic and intrinsic apoptosis signaling pathways with TRAIL and VEN showed strong synergistic antileukemic activity in AML cells with high levels of TP53INP2.
CONCLUSIONS: Our findings revealed that TP53INP2 is a predictor of responsiveness to TRAIL treatment and supported a potentially individualized therapeutic strategy for TP53INP2-positive AML patients.
METHODS: First, the relationship between TP53INP2 and the sensitivity of AML cells to TRAIL was determined by bioinformatics analysis of Cancer Cell Line Encyclopedia datasets, Cell Counting Kit-8 assays, flow cytometry (FCM) and cell line-derived xenograft (CDX) mouse models. Second, the mechanisms by which TP53INP2 participates in the response to TRAIL were analyzed by Western blot, ubiquitination, coimmunoprecipitation and immunofluorescence assays. Finally, the effect of TRAIL alone or in combination with the BCL-2 inhibitor venetoclax (VEN) on cell survival was explored using colony formation and FCM assays, and the effect on leukemogenesis was further investigated in a patient-derived xenograft (PDX) mouse model.
RESULTS: AML cells with high TP53INP2 expression were more sensitive to TRAIL in vitro and in vivo. Gain- and loss-of-function studies demonstrated that TP53INP2 significantly enhanced TRAIL-induced apoptosis, especially in AML cells with nucleophosmin 1 (NPM1) mutations. Mechanistically, cytoplasmic TP53INP2 maintained by mutant NPM1 functions as a scaffold bridging the ubiquitin ligase TRAF6 to caspase-8 (CASP 8), thereby promoting the ubiquitination and activation of the CASP 8 pathway. More importantly, simultaneously stimulating extrinsic and intrinsic apoptosis signaling pathways with TRAIL and VEN showed strong synergistic antileukemic activity in AML cells with high levels of TP53INP2.
CONCLUSIONS: Our findings revealed that TP53INP2 is a predictor of responsiveness to TRAIL treatment and supported a potentially individualized therapeutic strategy for TP53INP2-positive AML patients.
摘要：
背景：急性髓系白血病（AML）是一种预后不良的造血系统恶性肿瘤，尤其是老年AML患者。肿瘤坏死因子相关的凋亡诱导配体（TRAIL）被认为是一种有前途的抗癌药物，因为它选择性地诱导肿瘤细胞的外源性凋亡而不影响正常细胞。然而,临床试验表明,患者对TRAIL的反应是显著异质性的.有必要探索可预测的生物标志物，以预选对TRAIL具有更好反应性的AML患者。这里,我们研究了肿瘤蛋白p53诱导型核蛋白2(TP53INP2)在AML细胞对TRAIL治疗反应中的关键作用.
方法：首先，TP53INP2与AML细胞对TRAIL的敏感性之间的关系通过对癌细胞系百科全书数据集的生物信息学分析来确定，细胞计数试剂盒-8测定，流式细胞术（FCM）和细胞系来源的异种移植（CDX）小鼠模型。第二,通过蛋白质印迹分析TP53INP2参与TRAIL应答的机制，泛素化，共免疫沉淀和免疫荧光测定。最后,使用集落形成和FCM测定法探索TRAIL单独或与BCL-2抑制剂venetoclax（VEN）联合使用对细胞存活的影响，在患者来源的异种移植（PDX）小鼠模型中进一步研究了对白血病发生的影响。
结果：TP53INP2高表达的AML细胞在体外和体内对TRAIL更敏感。研究表明，TP53INP2显著增强TRAIL诱导的细胞凋亡，特别是在具有核磷蛋白1（NPM1）突变的AML细胞中。机械上,由突变体NPM1维持的细胞质TP53INP2充当桥接泛素连接酶TRAF6与caspase-8（CASP8）的支架，从而促进CASP8通路的泛素化和活化。更重要的是,用TRAIL和VEN同时刺激外源性和内源性凋亡信号通路,在TP53INP2高水平的AML细胞中显示出强的协同抗白血病活性.
结论：我们的研究结果表明，TP53INP2是TRAIL治疗反应性的预测因子，并支持TP53INP2阳性AML患者的潜在个体化治疗策略。
方法：首先，TP53INP2与AML细胞对TRAIL的敏感性之间的关系通过对癌细胞系百科全书数据集的生物信息学分析来确定，细胞计数试剂盒-8测定，流式细胞术（FCM）和细胞系来源的异种移植（CDX）小鼠模型。第二,通过蛋白质印迹分析TP53INP2参与TRAIL应答的机制，泛素化，共免疫沉淀和免疫荧光测定。最后,使用集落形成和FCM测定法探索TRAIL单独或与BCL-2抑制剂venetoclax（VEN）联合使用对细胞存活的影响，在患者来源的异种移植（PDX）小鼠模型中进一步研究了对白血病发生的影响。
结果：TP53INP2高表达的AML细胞在体外和体内对TRAIL更敏感。研究表明，TP53INP2显著增强TRAIL诱导的细胞凋亡，特别是在具有核磷蛋白1（NPM1）突变的AML细胞中。机械上,由突变体NPM1维持的细胞质TP53INP2充当桥接泛素连接酶TRAF6与caspase-8（CASP8）的支架，从而促进CASP8通路的泛素化和活化。更重要的是,用TRAIL和VEN同时刺激外源性和内源性凋亡信号通路,在TP53INP2高水平的AML细胞中显示出强的协同抗白血病活性.
结论：我们的研究结果表明，TP53INP2是TRAIL治疗反应性的预测因子，并支持TP53INP2阳性AML患者的潜在个体化治疗策略。
