PRMT1 inhibition promotes ferroptosis sensitivity via ACSL1 upregulation in acute myeloid leukemia

Acute myeloid leukemia (AML) is a hematological malignancy with an alarming mortality rate. The development of novel therapeutic targets or drugs for AML is urgently needed. Ferroptosis is a form of regulated cell death driven by iron-dependent lipid peroxidation. Recently, ferroptosis has emerged as a novel method for targeting cancer, including AML. Epigenetic dysregulation is a hallmark of AML, and a growing body of evidence suggests that ferroptosis is subject to epigenetic regulation. Here, we identified protein arginine methyltransferase 1 (PRMT1) as a ferroptosis regulator in AML. The type I PRMT inhibitor GSK3368715 promoted ferroptosis sensitivity in vitro and in vivo. Moreover, PRMT1-knockout cells exhibited significantly increased sensitivity to ferroptosis, suggesting that PRMT1 is the primary target of GSK3368715 in AML. Mechanistically, both GSK3368715 and PRMT1 knockout upregulated acyl-CoA synthetase long-chain family member 1 (ACSL1), which acts as a ferroptosis promoter by increasing lipid peroxidation. Knockout ACSL1 reduced the ferroptosis sensitivity of AML cells following GSK3368715 treatment. Additionally, the GSK3368715 treatment reduced the abundance of H4R3me2a, the main histone methylation modification mediated by PRMT1, in both genome-wide and ACSL1 promoter regions. Overall, our results demonstrated a previously unknown role of the PRMT1/ACSL1 axis in ferroptosis and suggested the potential value and applications of the combination of PRMT1 inhibitor and ferroptosis inducers in AML treatment.     

DNA甲基化靶向药物在急性髓系白血病中的应用

DNA甲基化和组蛋白甲基化是急性髓系白血病（acute myeloid leukemia,AML）表观遗传学调控的常见模式,针对甲基化过程的靶向治疗包括DNA甲基转移酶抑制剂、甲基化调节蛋白抑制剂及组蛋白甲基化调控蛋白抑制剂,其中DNA甲基转移酶（DNA methyltransferase,DNMT）抑制剂阿扎胞苷、地西他滨已上市进入临床,针对甲基化调节蛋白IDH1/2抑制剂也已进入Ⅱ期临床研究。此外,针对组蛋白甲基化调控蛋白EZH2、LSD1抑制剂也显示出良好体外抗白血病活性,部分已进入Ⅰ期临床研究,为AML的治疗提供新的选择。      

miR -148b 在急性髓系白血病患者及细胞系中的表达与生物学功能

目的：检测 miR -148b 在急性髓系白血病患者和细胞系中的表达水平。初步研究 miR -148b 在急性髓系白血病中的生物学功能。方法：收集急性髓系白血病患者56例,非恶性血液病对照组20例,提取骨髓有核细胞;培养 U937、Kasumi -1、THP -1三种 AML 细胞系。RT - PCR 检测 miR -148b 的表达水平。分别对U937细胞系及 Kasumi -1细胞系转染 miR -148b mimics 和 inhibitor。CCK -8法检测 U937细胞及 Kasumi -1细胞增殖情况,Annexin V/ PI 流式检测细胞凋亡,流式细胞术检测细胞周期。结果：miR -148b 在急性髓系白血病患者及细胞系中较非恶性血液病对照组表达明显下降。U937细胞转染 miR -148b mimics 后,增殖受抑制,细胞凋亡增加,细胞阻滞在 G0- G1期;Kasumi -1细胞转染 miR -148b inhibitor 后,增殖增加,细胞凋亡减少,G0- G1期细胞减少。结论：miR -148b 在急性髓系白血病患者及细胞系中的表达水平均较正常对照显著降低。miR -148b 在急性髓系白血病中起抑癌基因的作用,过表达 miR -148b 可以抑制细胞增殖,促进凋亡。     

Twist 1通过MPL促进髓系白血病细胞增殖和耐药

目的:检测Twist-1与骨髓增生性白血病病毒癌基因(myeloproliferative leukemia virus oncogene,MPL)在髓系白血病患者和髓系造血系统恶性肿瘤细胞系中表达的相关性,并探讨Twist-1是否通过MPL对髓系白血病细胞增殖、耐药发挥促进作用.方法:选取中国医学科学院血液病医院2005年1月至2008年12月初次诊断为急性髓系白血病(acute myeloid leu-kemia,AML)、慢性粒细胞白血病(chronic myeloid leukemia,CML)患者的骨髓标本41例(其中AML 23例、CML 18例),用Re-al-time PCR检测AML、CML患者以及髓系造血系统恶性肿瘤细胞系中Twist-1和MPL mRNA的表达情况,并分析其相关性.构建MPL过表达载体,制备慢病毒并感染髓系白血病细胞系K562、U937,通过细胞计数实验、集落形成实验以及药物敏感实验评价其对白血病细胞增殖、集落形成能力以及耐药的影响,并进一步确定Twist-1是否通过MPL发挥白血病促进作用.结果:在U937和K562细胞中过表达Twist-1明显增加MPL蛋白水平(P＜0.05),敲降Twist-1后MPL mRNA的表达水平明显下降(P＜0.01);AML、CML患者骨髓单核细胞(bone marrow mononuclear cells,BMMCs)中Twist-1 mRNA表达与MPL mRNA表达水平呈显著正相关(P＜0.05).过表达MPL使K562和U937细胞对化疗药物柔红霉素及伊马替尼的敏感性显著降低(P＜0.01),且提高K562-MPL、U937-MPL的细胞增殖、集落形成数目(均P＜0.01);干扰Twist-1并过表达MPL显著降低髓系白血病细胞增殖和集落形成(均P＜0.01).结论:Twist-1通过MPL促进AML、CML白血病细胞的增殖、存活和耐药.     

miR-148b在急性髓系白血病中的表达及与临床特征的关系

目的:检测miR-148b在急性髓系白血病(acute myeloid leukemia,AML)中的表达水平,分析其与临床特征的关系。方法:收集急性髓系白血病患者40例,非恶性血液病患者（对照组）20例,提取骨髓有核细胞,RT-PCR检测miR-148b的表达水平,分析其表达水平与临床特征的关系。结果:miR-148b在急性髓系白血病患者较对照组表达明显下降。miR-148b相对低表达组的患者较相对高表达组患者外周血白细胞计数及骨髓原始细胞比例较高。疗效比较中,完全缓解率及总生存率比较两组未见明显差异,但是miR-148b高表达组较低表达组复发率低（P=0.038）,无复发生存两组差异有统计学意义（P=0.029）。结论:miR-148b在急性髓系白血病患者中的表达水平较对照组显著降低。miR-148b的表达水平与AML临床特征具有一定的关联,对疾病复发具有重要意义。     

TWIST-1 promotes cell growth,drug resistance and progenitor clonogenic capacities in myeloid leukemia and is a novel poor prognostic factor in acute myeloid leukemia

Alterations of TWIST-1 expression are often seen in solid tumors and contribute to tumorigenesis and cancer progression. However, studies concerning its pathogenic role in leukemia are scarce. Our study shows that TWIST-1 is overexpressed in bone marrow mononuclear cells of patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Gain-of-function and loss-of-function analyses demonstrate that TWIST-1 promotes cell growth, colony formation and drug resistance of AML and CML cell lines. Furthermore, TWIST-1 is aberrantly highly expressed in CD34⁺CD38⁻ leukemia stem cell candidates and its expression declines with differentiation. Down-modulation of TWIST-1 in myeloid leukemia CD34⁺ cells impairs their colony-forming capacity. Mechanistically, c-MPL, which is highly expressed in myeloid leukemia cells and associated with poor prognosis, is identified as a TWIST-1 coexpressed gene in myeloid leukemia patients and partially contributes to TWIST-1-mediated leukemogenic effects. Moreover, patients with higher TWIST-1 expression have shorter overall and event-free survival (OS and EFS) in AML. Multivariate analysis further demonstrates that TWIST-1 overexpression is a novel independent unfavourable predictor for both OS and EFS in AML. These data highlight TWIST-1 as a new candidate gene contributing to leukemogenesis of myeloid leukemia, and propose possible new avenues for improving risk and treatment stratification in AML.     

m6A-RNA甲基化在正常造血和急性髓系白血病中的研究进展

m6A-RNA甲基化是一种类似于DNA甲基化或组蛋白修饰的表观遗传修饰方式。是由甲基化转移酶、去甲基转移酶和相关阅读蛋白共同调节的一种动态可逆的生物学过程,可以对 mRNA产生不同的生物学作用,包括mRNA剪切、出核、降解、影响mRNA稳定性和翻译效率等。越来越多的研究表明m6A-RNA甲基化在正常造血和急性髓系白血病中发挥重要的调控作用。本文就m6A-RNA甲基化在正常造血和AML中的研究进展作一综述,旨在从表观转录组学层面深入了解AML的发病机制,为探索AML靶向治疗药物提供一种新的研究策略。     

Combined testing for CCAAT/enhancer-binding protein alpha (CEBPA) mutations and promoter methylation in acute myeloid leukemia demonstrates shared phenotypic features

Loss of function mutations in CCAAT/enhancer binding protein alpha (CEBPA) have been identified in acute myeloid leukemia (AML) and bi-allelic (double) CEBPA mutations are associated with improved prognosis in cases of cytogenetically normal-AML. In a subset of AML patients lacking CEBPA mutations, core promotor methylation of CEBPA has been described and is associated with a gene expression profile similar to the mutated cases including the expression of T cell associated genes such as CD7. However, the overall incidence and pattern of CEBPA mutations and core promoter methylation has not been thoroughly explored in a larger subset of AML with expression of CD7. Here we describe a simple and clinically deployable CEBPA promoter methylation test and the results of combined testing for CEBPA mutations and promoter methylation in 102 cases of AML, including 43 CD7+ cases. Overall, there were 5 methylated cases, 6 cases with double mutations, and 3 cases with single mutations. Significantly, 10 of 43 CD7+ cases (23%) had either methylated or double-mutated CEBPA. The CD7+ subset included all 5 methylated cases and 5 of the 6 cases with double mutations. All 3 cases with single mutations were CD7−. No case exhibited both hypermethylation and mutations. We find that promoter methylation accounts for half of those CD7+ cases with CEBPA dysregulating abnormalities. Furthermore, methylated cases and those with bi-allelic CEBPA mutations have similar phenotypic features including expression of CD7 and lack of co-incident NPM1 mutations. Our study suggests that methylation testing may be as important as mutation testing for identifying AML cases with CEBPA dysregulation and may be indicated in the routine prognostic workup of AML.     

Linc00355在结肠癌中表达上调并促进肿瘤细胞增殖和侵袭

目的:探讨长链非编码RNA (long non-coding RNA,lncRNA) Linc00355在结肠癌组织中的表达水平,以及沉默Linc00355表达对结肠癌HCT116细胞增殖及迁移和侵袭能力的影响.方法:通过对肿瘤基因组图谱(The Cancer Genome Atlas,TCGA)和基因表达综合(Gen...     

miR-21-5p promotes cell proliferation by targeting BCL11B in Thp-1 cells

Acute myeloid leukemia (AML) is a highly heterogeneous disease that remains untreatable. MicroRNAs (miRNAs or miRs) play important roles in the pathogenesis of leukemia. miR-21 is highly expressed in multiple types of human cancer and displays oncogenic activities; however, the clinical significance of miR-21 in AML remains unclear. In the present study, it was demonstrated that miR-21 levels were high in patients with AML and in AML cell lines. Further experiments demonstrated that overexpression of miR-21 in Thp-1 human monocytes derived from acute mononuclear leukemia peripheral blood promoted cell proliferation, while downregulation of miR-21-5p, a mature sequence derived from the 5′ end of the miR-21 stem-loop precursor (another mature sequence, miR-21-3p, is derived form 3′ end of miR-21), inhibited cell proliferation. Specifically, it was observed that overexpression of miR-21 could promote the transition of Thp-1 cells into the S and G2/M phases of the cell cycle, as shown by flow cytometry. Furthermore, inhibition of miR-21-5p arrested cells in the S and G2/M phases. Finally, BCL11B was determined to be a functional target of miR-21-5p by luciferase assays. Our study revealed functional and mechanistic associations between miR-21 and BCL11B in Thp-1 cells, which could serve to guide clinical treatment of AML.     

沉默SIRT2基因对急性粒细胞白血病细胞增殖的影响

目的 SIRT2是组蛋白去乙酰化酶家族成员之一,其参与多种肿瘤的发生和发展.本研究旨在探讨SIRT2基因在人急性粒细胞白血病细胞NB4和耐药细胞HL60/A增殖中的作用,分析SIRT2对于细胞周期的影响,寻找治疗急性粒细胞白血病的新靶点.方法 蛋白质印迹法检测SIRT2在急性粒细胞白血病细胞HL60/A和NB4中的表达,针对SIRT2 mRNA不同的靶序列设计并合成shRNA,构建真核表达干扰载体,制备慢病毒并感染HL60/A和NB4细胞,用嘌呤霉素筛选出稳定表达shRNA的细胞,应用蛋白质印迹法验证干扰效率;采用改良MTT法检测沉默SIRT2基因前后HL60/A和NB4细胞增殖的改变;用流式细胞术检测沉默SIRT2基因前后细胞周期的变化.结果 在HL60/A和NB4细胞中SIRT2蛋白表达水平均增高,F=43.22,P=0.002.用慢病毒感染HL60/A(F=184.9,P＜0.001)和NB4(F=354.7,P＜0.001)后,SIRT2基因的蛋白表达水平显著降低.MTT结果显示,与对照组细胞比较,48 h沉默SIRT2基因的HL60/A(F=208.4,P＜0.001)和NB4(F=27.58,P＜0.001)细胞的生长速度变慢;于72 h这种抑制作用更加明显,其中HL60/A的增殖速度明显变慢,F=555.9,P＜0.001.流式细胞术检测细胞周期结果显示,G0～G1期百分比,与对照组HL60/A/con(27.62±1.01)％相比,干扰组HL60/A/sh1 (37.14±0.03)％(F=7.61,P＜0.001)和HL60/sh2(37.51±3.63)％(F=3.89,P=0.009)比例显著增加;与对照组NB4/con(27.49±0.50)％相比,干扰组NB4/sh1(38.26±1.93)％(F=8.02,P=0.007)和NB4/sh2(35.23±1.11)％(F=3.50,P＜0.001)比例显著增加;S期细胞百分比,与对照组HL60/A/con(51.94±1.15)％相比,干扰组HL60/A/sh1 (40.24±0.66)％(F=3.34,P＜0.001)和HL60/sh2(42.73±2.76)％(F=10.33,P=0.004)比例显著降低;与对照组NB4/con(50.65±0.23)％相比,干扰组NB4/sh1(39.58±1.11)％(F=3.02,P＜0.001)和NB4/sh2(43.82±1.33)％(F=10.42,P=0.011)比例显著降低;G2～M期细胞比例无明显变化.结论 干扰SIRT2基因的表达可使细胞周期阻滞在G0～G1期,从而抑制急性粒细胞白血病细胞HL60/A和NB4的增殖.