Original articles

Study on mechanism of synergistic effect of ASP2215 combined with SAHA on FLT3-ITD mutant cell line

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  • Department of Hematology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China

Received date: 2018-08-25

  Online published: 2018-10-25

Abstract

Objective: To study the synergistic effect and mechanism of FLT3 inhibitor ASP2215 combined with HDAC inhibitor SAHA on FLT3-ITD mutated cell line MV4-11. Methods: MV4-11cells were treated with ASP2215, SAHA or ASP2215 combined with SAHA at different concentrations, and then cell morphological changes were observed, cell viability was detected by CCK-8 method and apoptosis rate measured by flow cytometry. Phosphorylation of FLT3 and downstream signaling molecule STAT5, as well as the levels of apoptosis regulated proteins Mcl-1, Bcl-xL, Bcl-2, Baxand Caspase-9/3 were detected by Western blot. Results: ① Compared with wild-type FLT3 cell line THP-1, ASP2215 could specifically inhibit the proliferation of FLT3-ITD mutated AML cell line MV4-11. ② ASP2215 or SAHA alone could inhibit the viability of MV4-11 cells in a dose and time dependent manner. Moreover, the combination of the two drugs could synergistically inhibit the viability of MV4-11cells, and the CI values of combination of the two drugs at different concentrationswere all less than 1. ③ASP2215 or SAHA alone could induce apoptosis of MV4-11 cells in a dose and time dependent manner, and there was a synergistic effect for the two drugs combined to induce apoptosis of MV4-11 cells. Apoptosis was accompanied by cleaved activation of caspase-3 and caspase-9. Combination of the two drugs caused more cleaved activation of caspase-3 and caspase-9 than the effect of single-drug. Morphologically, the changes of apoptosis and necrosis increased with the increase of drug concentration, and combination of the two drugs could lead to more obvious cell apoptosis and necrosis changes. ④The FLT3 inhibitor ASP2215 could reduce the phosphorylation level of FLT3 receptor and the downstream molecule STAT5, and SAHA also had a slight inhibitory effect on the phosphorylation of FLT3 and the downstream molecule STAT5. Both ASP2215 and SAHA induced a decrease in McL-1 and Bcl-xL anti-apoptotic proteins, with a slight down-regulation of Bcl-2 protein and a slight up-regulation of Bax protein expression. Combination of the two drugs further reduced the Mcl-1, Bcl-xL expression level and Bcl-2/Bax protein ratio when compared with the effect of single-drug. Conclusions: ASP2215 combined with SAHA can synergistically inhibit the proliferation and enhance the apoptosis of MV4-11 cell line with FLT3-ITD mutation. The mechanism involves the inhibition of FLT3-STAT5 pathway and the regulation of apoptosis-related proteins Mcl-1, Bcl-xL and Bcl-2/Bax protein ratio.

Cite this article

ZHU Qingfeng, HU Xiaoli, ZHU Jianyi, LANG Wenjing, ZHONG Jihua, CHEN Fangyuan . Study on mechanism of synergistic effect of ASP2215 combined with SAHA on FLT3-ITD mutant cell line[J]. Journal of Diagnostics Concepts & Practice, 2018 , 17(05) : 538 -546 . DOI: 10.16150/j.1671-2870.2018.05.011

References

[1] Döhner H, Weisdorf DJ, Bloomfield CD.Acute Myeloid Leukemia[J]. N Engl J Med,2015,373(12):1136-1152.
[2] Lagunas-Rangel FA, Chávez-Valencia V.FLT3-ITD and its current role in acute myeloid leukaemia[J]. Med Oncol,2017,34(6):114.
[3] Takahashi S.Downstream molecular pathways of FLT3 in the pathogenesis of acute myeloid leukemia: biology and therapeutic implications[J]. J Hematol Oncol,2011,4:13.
[4] Smith CC, Wang Q, Chin CS, et al.Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia[J]. Nature,2012,485(7397):260-263.
[5] Lee LY, Hernandez D, Rajkhowa T, et al.Preclinical studies of gilteritinib, a next-generation FLT3 inhibitor[J]. Blood,2017,129(2):257-260.
[6] Mori M, Kaneko N, Ueno Y, et al.Gilteritinib, a FLT3/AXL inhibitor, shows antileukemic activity in mouse models of FLT3 mutated acute myeloid leukemia[J]. Invest New Drugs,2017,35(5):556-565.
[7] Perl AE, Altman JK, Cortes J, et al.Selective inhibition of FLT3 by gilteritinib in relapsed or refractory acute myeloid leukaemia: a multicentre, first-in-human, open-label, phase 1-2 study[J]. Lancet Oncol,2017,18(8):1061-1075.
[8] Lin WH, Yeh TK, Jiaang WT, et al.Evaluation of the antitumor effects of BPR1J-340, a potent and selective FLT3 inhibitor, alone or in combination with an HDAC inhibitor, vorinostat, in AML cancer[J]. PLoS One,2014, 9(1):e83160.
[9] Stahl M, Gore SD, Vey N, et al.Lost in translation? Ten years of development of histone deacetylase inhibitors in acute myeloid leukemia and myelodysplastic syndromes[J]. Expert Opin Investig Drugs,2016,25(3):307-317.
[10] Richon VM, Garcia-Vargas J, Hardwick JS.Development of vorinostat: current applications and future perspectives for cancer therapy[J]. Cancer Lett,2009,280(2):201-210.
[11] Siegel D, Hussein M, Belani C, et al.Vorinostat in solid and hematologic malignancies[J]. J Hematol Oncol,2009, 2:31.
[12] Zhou J, Bi C, Chng WJ, et al.PRL-3, a metastasis asso-ciated tyrosine phosphatase, is involved in FLT3-ITD signaling and implicated in anti-AML therapy[J]. PLoS One,2011,6(5):e19798.
[13] Choudhary C, Brandts C, Schwable J, et al.Activation mechanisms of STAT5 by oncogenic Flt3-ITD[J]. Blood,2007,110(1):370-374.
[14] Juen L, Brachet-Botineau M, Parmenon C, et al.New Inhibitor Targeting Signal Transducer and Activator of Transcription 5 (STAT5) Signaling in Myeloid Leukemias[J]. J Med Chem,2017,60(14):6119-6136.
[15] Nishioka C, Ikezoe T, Yang J, et al.MS-275, a novel histone deacetylase inhibitor with selectivity against HDAC1, induces degradation of FLT3 via inhibition of chaperone function of heat shock protein 90 in AML cells[J]. Leuk Res,2008,32(9):1382-1392.
[16] 王卫东, 陈正堂. Bcl-2/Bax比率与细胞“命运”[J]. 中国肿瘤生物治疗杂志,2007,14(4):393-396.
[17] Warr MR, Shore GC.Unique biology of Mcl-1: therapeutic opportunities in cancer[J]. Curr Mol Med,2008,8(2):138-147.
[18] Yoshimoto G, Miyamoto T, Jabbarzadeh-Tabrizi S, et al.FLT3-ITD up-regulates MCL-1 to promote survival of stem cells in acute myeloid leukemia via FLT3-ITD-specific STAT5 activation[J]. Blood,2009,114(24):5034-5043.
[19] Rosato RR, Almenara JA, Kolla SS, et al.Mechanism and functional role of XIAP and Mcl-1 down-regulation in flavopiridol/vorinostat antileukemic interactions[J]. Mol Cancer Ther,2007,6(2):692-702.
[20] Gesbert F, Griffin JD.Bcr/Abl activates transcription of the Bcl-X gene through STAT5[J]. Blood,2000,96(6):2269-2276.
[21] Horita M, Andreu EJ, Benito A, et al.Blockade of the Bcr-Abl kinase activity induces apoptosis of chronic myelogenous leukemia cells by suppressing signal transducer and activator of transcription 5-dependent expression of Bcl-xL[J]. J Exp Med,2000,191(6):977-984.
[22] Minami Y, Yamamoto K, Kiyoi H, et al.Different antiapoptotic pathways between wild-type and mutated FLT3: insights into therapeutic targets in leukemia[J]. Blood,2003,102(8):2969-2975.
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