靶向干预SETDB1对急性髓系白血病的治疗作用

Therapeutic Effects of Targeted Intervention of SETDB1 in Acute Myeloid Leukemia

关键词:  SETDB1  急性髓系白血病  恶性进展  分化治疗

Key words:  SETDB1  acute myeloid leukemia  malignant progression  differentiation therapy

摘要:

目的 探究组蛋白甲基转移酶SETDB1在急性髓系白血病(acute myeloid leukemia，AML)恶性进展中的作用及干扰SETDB1对AML的治疗效果。方法 基于TCGA、GTEx、TARGET数据库，分析SETDB1在不同肿瘤的表达情况，并对比分析在AML骨髓细胞和正常骨髓细胞中的表达情况，进一步分析SETDB1的表达与AML患者不同风险分层、不同FAB(French-American-British)分型及微小残留病灶(minimal residual disease，MRD)的相关性；对SETDB1高/低表达患者进行基因富集分析。通过RT-qPCR考察shRNA序列对SETDB1的沉默效率；通过台盼蓝染色法考察沉默SETDB1对AML细胞的增殖及活力的影响；通过检测细胞表面抗原CD11b、CD14的比例、细胞核形态和氯化硝基四氮唑蓝(NBT)还原能力考察沉默SETDB1对AML细胞的分化治疗作用。结果 与其他肿瘤相比，SETDB1在AML中显著高表达，且在AML中的表达水平显著高于正常骨髓细胞。SETDB1在AML高风险患者中、MRD残留患者中也显著高表达。沉默SETDB1可显著抑制AML细胞的增殖能力，并对其细胞活力没有显著影响。此外，SETDB1高表达的AML患者存在造血干细胞相关基因和分化阻碍相关基因的显著富集，以及SETDB1在未分化或者部分分化的M0~M2亚型中相对高表达。更为重要的是，沉默SETDB1可显著诱导AML细胞分化，表现为增加细胞表面分化标志物CD11b、CD14的表达、细胞核形态从圆形或椭圆形向马蹄形的分化状态转变以及细胞NBT还原能力显著增加。结论 AML中特异性高表达的SETDB1与AML恶性进展密切相关，靶向SETDB1有望成为AML分化治疗的潜在策略。

Abstract:

OBJECTIVE Acute myeloid leukemia(AML) is an aggressive hematological malignancy. Despite remarkable insights into the molecular pathogenesis and albeit modest advances in treatment, the 5-year survival rate of AML patients is only 25%-30%. A characteristic feature of AML is failure to terminally differentiate into functional mature myeloid cells. A notable exception in AML therapy has been use of all-trans retinoic acid(ATRA) in individuals with acute promyelocytic leukemia (APL). ATRA can induce APL cell differentiation, highlighting that differentiation-based therapy holds great promise for AML treatment. Unfortunately, ATRA only reverses the survival curve of patients with APL, which only accounts for 10% of the total AML. Therefore, there is an unmet need to reveal new druggable targets for broad differentiation-based therapies. SET domain, bifurcated 1(SETDB1) is a histone 3 lysine 9(H3K9)-specific methyltransferase and is essential for the gene expression via regulating histone methylation. Aberrant expression of epigenetic regulators often leads to myeloid malignancies, such as histone demethylase LSD1, METTL3, indicated targeting epigenetic regulators in AML leukemia treatment holds great promise. SETDB1 is involved in regulating cancer development, such as abnormally high expression in hepatocellular carcinoma(HCC) and knocking down SETDB1 expression inhibits the motility and metastatic ability of HCC. However, it is still unclear about the role of SETDB1 in AML leukemogenesis and malignant progression, and whether targeted intervention of SETDB1 can be used for leukemia differentiation therapy. Therefore, this study is aim to explore the role of histone methyltransferase SETDB1 in the malignant progression of AML and the therapeutic effect of SETDB1 suppression in AML. METHODS Based on TCGA(The Cancer Genome Atlas) and GTEx(Genotype-Tissue Expression) databases, an analysis of the expression of SETDB1 in different cancer species and a comparison of the expression of SETDB1 in bone marrow cells of AML patients and normal bone marrow cells was conducted. Additionally, the correlation between the expression of SETDB1 and different risk stratification, micro residual disease(MRD) as well as different FAB(French-American-British) subtypes in AML patients were analyzed based on information obtained from the TCGA and TARGET databases. GSEA(Gene set enrichment analysis) for patients with high and low SETDB1 expression were performed to analysis the relationship between SETDB1 expression and differentiation. During the research, the silencing efficiency of SETDB1 was examined by RT-qRCR in AML cells. Furthermore, the effect of SETDB1 knockdown on the proliferation and viability of AML cells was evaluated using Trypan blue staining assay. CD11b, CD14 expression level alongside cell nuclear morphologic changes and nitrotetrazolium blue chloride(NBT) reduction ability were used to determine the therapeutic effect of shSETDB1 in AML cell differentiation. RESULTS SETDB1 was significantly highly expressed in AML patients compared to other cancer types, and was also significantly higher expressed than that in normal bone marrow cells. SETDB1 was obviously highly expressed in high-risk AML patients as well as patients with residual MRD. SETDB1 silencing significantly inhibited AML cell proliferation without any obvious cell death. Besides, genes typically upregulated in hematopoietic stem cells(HSCs) and downregulated in myeloid cell development were highly enriched in patients with high SETDB1 expression and SETDB1 was also found to be relatively highly expressed in undifferentiated or partially differentiated M0-M2 FAB subtypes. More importantly, flow cytometry analysis showed that shSETDB1 significantly increased the expression of CD11b and CD14 in AML cells and the upregulation ratio of CD11b and CD14 corresponded to the silencing effect of SETDB1. Moreover, SETDB1-declined cells had a more mature nuclear morphology and enhanced NBT-reducing ability in AML cells, suggesting the depletion of SETDB1 may drive myeloid differentiation and maturation in AML cells. CONCLUSION Histone methyltransferase SETDB1 is specifically highly expressed in AML and closely associates with the malignant progression of leukemia. Targeting SETDB1 is expected to be a promising strategy for differentiation therapy in AML, which provides new opportunities for AML treatment.