上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2022 , Vol. 42 >Issue 10: 1383 - 1393

DOI: https://doi.org/10.3969/j.issn.1674-8115.2022.10.003

论著 · 基础研究

去泛素化酶UCHL3维持FLT3-ITD阳性急性髓性白血病细胞存活的功能研究

  • 胡家诚 ,
  • 朱倩 ,
  • 王佳琦 ,
  • 吴英理 ,
  • 雷虎
展开
  • 上海交通大学基础医学院病理生理学系，上海 200025
胡家诚（2000—），男，本科生；电子信箱：2871416798@qq.com。
吴英理，电子信箱：wuyingli@shsmu.edu.cn。
雷 虎，电子信箱：hulei@shsmu.edu.cn

收稿日期: 2022-04-04

  录用日期: 2022-09-16

  网络出版日期: 2022-12-02

基金资助

国家自然科学基金(82170145);上海市高水平地方高校创新团队(SHSMU-ZDCX20211802)

收起

Function of UCHL3 in maintaining the survival of FLT3-ITD positive acute myeloid leukemia cells

  • Jiacheng HU ,
  • Qian ZHU ,
  • Jiaqi WANG ,
  • Yingli WU ,
  • Hu LEI
Expand
  • Department of Pathophysiology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
WU Yingli, E-mail: wuyingli@shsmu.edu.cn.
LEI Hu, E-mail: hulei@shsmu.edu.cn

Received date: 2022-04-04

  Accepted date: 2022-09-16

  Online published: 2022-12-02

Supported by

National Natural Science Foundation of China(82170145);Innovative Research Team of High-Level Local Universities in Shanghai(SHSMU-ZDCX20211802)

Fold

摘要

目的·探究FMS样的酪氨酸激酶3（FMS-like tyrosine kinase 3，FLT3）近膜区的内部串联重复（FLT3-ITD）阳性急性髓系白血病（acute myeloid leukemia，AML）细胞中存在表达差异的去泛素化酶及其对FLT3-ITD阳性AML细胞功能的影响。方法·采用Western blotting检测外源性导入FLT3-ITD质粒和使用FLT3-ITD抑制剂对部分去泛素化酶蛋白表达量的影响；利用癌症基因组图谱（the cancer genome atlas，TCGA）公共数据库分析泛素羧基末端水解酶L3（ubiquitin carboxyl-terminal hydrolase L3，UCHL3）在FLT3-ITD阳性患者中的表达情况及与AML患者预后的相关性；利用短发夹RNA（short hairpin RNA，shRNA）技术在FLT3-ITD阳性AML细胞系中敲低所筛选出的去泛素化酶；采用细胞计数、CCK-8法、流式细胞术、瑞氏染色检测所筛选出的去泛素化酶对AML细胞功能的影响以及敲低相应的去泛素化酶或使用其抑制剂与全反式维甲酸（all-trans-retinoic acid，ATRA）的合加效应。结果·Ba/F3细胞中表达FLT3-ITD后能够显著诱导UCHL3的表达，而在FLT3-ITD阳性的MOLM-13和MV4-11细胞系中抑制FLT3-ITD的活性后能够显著抑制UCHL3的表达，该抑制作用具有时间和浓度依赖性。利用数据库分析发现FLT3的表达与UCHL3的表达量在AML中具有显著正相关性（P=0.000），并且UCHL3表达量与AML患者生存率存在显著负相关性（P=0.016）。敲低UCHL3对FLT3-ITD阳性AML细胞有抑制增殖和促进凋亡的作用，且敲低UCHL3后细胞核型发生显著改变，分化相关转录因子PU.1和C/EBPβ表达显著升高。UCHL3敲低显著增强了100 nmol/L ATRA诱导的MOLM-13和MV4-11细胞凋亡，并且相应的凋亡相关蛋白PARP1、caspase 9和caspase 3均发生明显剪切。同时，敲低UCHL3的细胞在ATRA处理后出现更多核体积变小、核凹陷、呈杆状或不规则状的细胞特征。ATRA与UCHL3抑制剂TCID合加的量效反应曲线表明，TCID与ATRA协同能够抑制MOLM-13和MV4-11细胞活率。结论·FLT3-ITD上调UCHL3促进AML细胞的存活，UCHL3与AML患者预后负相关；敲低或抑制UCHL3协同ATRA能够抑制FLT3-ITD阳性AML细胞的存活。

关键词： 泛素羧基末端水解酶L3; 急性髓系白血病; FMS样的酪氨酸激酶3; FMS样的酪氨酸激酶3近膜区的内部串联重复; 全反式维甲酸

本文引用格式

胡家诚 , 朱倩 , 王佳琦 , 吴英理 , 雷虎 . 去泛素化酶UCHL3维持FLT3-ITD阳性急性髓性白血病细胞存活的功能研究[J]. 上海交通大学学报（医学版）, 2022 , 42(10) : 1383 -1393 . DOI: 10.3969/j.issn.1674-8115.2022.10.003

Abstract

Objective ·To explore differently expressed deubiquitinases in acute myeloid leukemia (AML) cells with internal tandem duplications of the FLT3 (FLT3-ITD) gene mutation. Methods ·The expressions of some deubiquitinases were detected by Western blotting after transforming exogenous FLT3-ITD or the use of FLT3-ITD inhibitor. The Cancer Genome Atlas (TCGA) public database was used to analyze the expression of UCHL3 in FLT3-ITD-positive patients and its correlation with the prognosis of AML patients. Certain deubiquitinase was knocked down by short hairpin RNA (shRNA) in AML cell lines. Cell counting, CCK-8 assay, flow cytometry and wright stain were used to detect the effect of certain deubiquitinase on the function of AML cells and the combined effect of knockdown of corresponding deubiquitinase or using its inhibitor with all-trans-retinoic acid (ATRA). Results ·Expression of FLT3-ITD in Ba/F3 cells significantly induced the protein expression of UCHL3, while inhibition of FLT3-ITD activity in MOLM-13 and MV4-11 cell lines significantly inhibited the expression of UCHL3 in a time- and concentration-dependent manner. TCGA database analysis showed that there was a significant positive correlation between the expression of FLT3 and the expression of UCHL3 in AML (P=0.000), and a significant negative correlation between the expression of UCHL3 and the survival rate of AML patients (P=0.016). UCHL3 knockdown inhibited proliferation and promoted apoptosis of MOLM-13 and MV4-11 cells, and the expression of differentiation-related transcription factors PU.1 and C/EBPβ were significantly increased. Knockdown of UCHL3 significantly enhanced the apoptosis of MOLM-13 and MV4-11 cells induced by 100 nmol/L ATRA, and the apoptosis-related caspase proteins PARP1, caspase 9 and caspase 3 were significantly cleaved. Meanwhile, UCHL3 knockdown cells showed more features of small nuclear volume, nuclear depression, and rod-shaped or irregular cells after ATRA treatment. The dose-response curves of ATRA and TCID detected by CCK-8 assay showed that UCHL3 inhibitor TCID and ATRA synergistic inhibited FLT3-ITD-positive AML cells survival. Conclusion ·FLT3-ITD up-regulates UCHL3 expression and promotes the survival of AML cells. UCHL3 is negatively correlated with the prognosis of AML patients. Knockdown or inhibition of UCHL3 combines with ATRA to inhibit the survival of FLT3-ITD-positive AML cells.

Key words： ubiquitin carboxyl-terminal hydrolase L3 (UCHL3); acute myeloid leukemia (AML); FMS-like tyrosine kinase 3 (FLT3); FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD); all-trans retinoic acid (ATRA)

参考文献

1 DE KOUCHKOVSKY I, ABDUL-HAY M. Acute myeloid leukemia: a comprehensive review and 2016 update[J]. Blood Cancer J, 2016, 6(7): e441.
2 SHORT N J, RYTTING M E, CORTES J E. Acute myeloid leukaemia[J]. Lancet, 2018, 392(10147): 593-606.
3 ANTAR A I, OTROCK Z K, JABBOUR E, et al. FLT3 inhibitors in acute myeloid leukemia: ten frequently asked questions[J]. Leukemia, 2020, 34(3): 682-696.
4 WU M, LI C T, ZHU X P. FLT3 inhibitors in acute myeloid leukemia[J]. J Hematol Oncol, 2018, 11(1): 133.
5 MOREAU P, RICHARDSON P G, CAVO M, et al. Proteasome inhibitors in multiple myeloma: 10 years later[J]. Blood, 2012, 120(5): 947-959.
6 MANASANCH E E, ORLOWSKI R Z. Proteasome inhibitors in cancer therapy[J]. Nat Rev Clin Oncol, 2017, 14(7): 417-433.
7 REINCKE M, SBIERA S, HAYAKAWA A, et al. Mutations in the deubiquitinase gene USP8 cause Cushing's disease[J]. Nat Genet, 2015, 47(1): 31-38.
8 WILLIAMS S A, MAECKER H L, FRENCH D M, et al. USP1 deubiquitinates ID proteins to preserve a mesenchymal stem cell program in osteosarcoma[J]. Cell, 2011, 146(6): 918-930.
9 MENNERICH D, KUBAICHUK K, KIETZMANN T. DUBs, hypoxia, and cancer[J]. Trends Cancer, 2019, 5(10): 632-653.
10 HE M J, ZHOU Z, WU G, et al. Emerging role of DUBs in tumor metastasis and apoptosis: therapeutic implication[J]. Pharmacol Ther, 2017, 177: 96-107.
11 AKIYAMA H, UMEZAWA Y, ISHIDA S, et al. Inhibition of USP9X induces apoptosis in FLT3-ITD-positive AML cells cooperatively by inhibiting the mutant kinase through aggresomal translocation and inducing oxidative stress[J]. Cancer Lett, 2019, 453: 84-94.
12 WEISBERG E L, SCHAUER N J, YANG J, et al. Inhibition of USP10 induces degradation of oncogenic FLT3[J]. Nat Chem Biol, 2017, 13(12): 1207-1215.
13 SCHAUER N J, MAGIN R S, LIU X X, et al. Advances in discovering deubiquitinating enzyme (DUB) inhibitors[J]. J Med Chem, 2020, 63(6): 2731-2750.
14 OUYANG L L, YAN B, LIU Y T, et al. The deubiquitylase UCHL3 maintains cancer stem-like properties by stabilizing the aryl hydrocarbon receptor[J]. Signal Transduct Target Ther, 2020, 5(1): 78.
15 SONG Z W, LI J H, ZHANG L, et al. UCHL3 promotes pancreatic cancer progression and chemo-resistance through FOXM1 stabilization[J]. Am J Cancer Res, 2019, 9(9): 1970-1981.
16 LI J N, ZHENG Y, LI X F, et al. UCHL3 promotes proliferation of colorectal cancer cells by regulating SOX12 via AKT/mTOR signaling pathway[J]. Am J Transl Res, 2020, 12(10): 6445-6454.
17 SATO A, KAMIO N, YOKOTA A, et al. C/EBPβ isoforms sequentially regulate regenerating mouse hematopoietic stem/progenitor cells[J]. Blood Adv, 2020, 4(14): 3343-3356.
18 SCOTT E W, SIMON M C, ANASTASI J, et al. Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages[J]. Science, 1994, 265(5178): 1573-1577.
19 MUELLER B U, PABST T, FOS J, et al. ATRA resolves the differentiation block in t(15;17) acute myeloid leukemia by restoring PU.1 expression[J]. Blood, 2006, 107(8): 3330-3338.
20 KHAN I, HALASI M, PATEL A, et al. FOXM1 contributes to treatment failure in acute myeloid leukemia[J]. JCI Insight, 2018, 3(15): e121583.
21 LIU LL, ZHANG D H, MAO X, et al. Over-expression of FoxM1 is associated with adverse prognosis and FLT3-ITD in acute myeloid leukemia[J]. Biochem Biophys Res Commun, 2014, 446(1): 280-285.
22 SHENG Y, YU C J, LIU Y, et al. FOXM1 regulates leukemia stem cell quiescence and survival in MLL-rearranged AML[J]. Nat Commun, 2020, 11(1): 928.
23 KHAN I, HALASI M, ZIA M F, et al. Nuclear FOXM1 drives chemoresistance in AML[J]. Leukemia, 2017, 31(1): 251-255.
24 SONG Z W, TU X Y, ZHOU Q, et al. A novel UCHL3 inhibitor, perifosine, enhances PARP inhibitor cytotoxicity through inhibition of homologous recombination-mediated DNA double strand break repair[J]. Cell Death Dis, 2019, 10(6): 398.
25 WANG Z Y, CHEN Z. Acute promyelocytic leukemia: from highly fatal to highly curable[J]. Blood, 2008, 111(5): 2505-2515.
26 SCHOLL S, MüLLER R, CLEMENT J H, et al. ATRA can enhance apoptosis that is induced by Flt3 tyrosine kinase inhibition in Flt3-ITD positive cells[J]. Leuk Res, 2006, 30(5): 633-642.
27 CHI H T, LY B T K, VU H A, et al. Synergistic effect of all?trans retinoic acid in combination with protein kinase C412 in FMS-like tyrosine kinase 3-mutated acute myeloid leukemia cells[J]. Mol Med Rep, 2015, 11(5): 3969-3975.
Options
文章导航

/

〈 〉