Tetrahydrobenzimidazole TMQ0153 triggers apoptosis, autophagy and necroptosis crosstalk in chronic myeloid leukemia (Part 3 Figure 5-9, Supple 1-12))

Abstract
Chronic myeloid leukemia (CML) results from a t(9;22) (q34; q11) translocation, also called Philadelphia chromosome (Ph). This reciprocal translocation causes a constitutively-activated tyrosine kinase BCR-ABL fusion gene. By comparing imatinib-sensitive and -resistant CML cell models, we investigated the molecular mechanisms by which tetrahydrobenzimidazole derivative TMQ0153 triggered caspase-dependent apoptosis at low concentrations accompanied by loss of mitochondrial membrane potential (MMP) and increase of cytosolic free Ca2+ levels. Interestingly, at higher concentrations, TMQ0153 induced necroptotic cell death with accumulation of ROS, both preventable by N-acetyl-L-cysteine (NAC) pretreatment. At necroptosis-inducing concentrations, we observed increased ROS and decreased ATP and GSH levels, concomitant with protective autophagy induction. Inhibitors such as bafilomycin A1 (baf-A1) and siRNA against beclin 1 abrogated autophagy, sensitized CML cells against TMQ0153 and enhanced necroptotic cell death. Importantly, TMQ153-induced necrosis led to cell surface exposure of calreticulin (CRT) and ERp57 as well as the release of extracellular ATP and high mobility group box (HMGB1) demonstrating the immunological cell death markers by TMQ0153. We validated the anti-cancer potential of TMQ0153 by in vivo inhibition of K562 microtumor formation in zebrafish. Taken together, our findings provide evidence that cellular stress and redox modulation by TMQ0153 concentration-dependently leads to different cell death modalities including controlled necrosis in CML cell models.

摘要
慢性髓系白血病（Chronic myeloid leukemia, CML）由t(9;22)(q34;q11)易位引发，该易位又称费城染色体（Philadelphia chromosome, Ph）。这种相互易位会导致组成型激活的酪氨酸激酶BCR-ABL融合基因的产生。本研究通过对比伊马替尼敏感及耐药的慢性髓系白血病细胞模型，探究了四氢苯并咪唑类衍生物TMQ0153在低浓度下，通过伴随线粒体膜电位（mitochondrial membrane potential, MMP）丧失与胞浆游离钙离子水平升高，触发半胱天冬酶依赖性凋亡的分子机制。有趣的是，在高浓度下，TMQ0153可诱导伴有活性氧（Reactive Oxygen Species, ROS）积累的坏死性凋亡细胞死亡，该效应可通过N-乙酰-L-半胱氨酸（N-acetyl-L-cysteine, NAC）预处理予以阻断。在坏死性凋亡诱导浓度下，我们观察到ROS水平升高、三磷酸腺苷（ATP）与谷胱甘肽（GSH）水平降低，同时伴随保护性自噬的诱导。巴弗洛霉素A1（bafilomycin A1, baf-A1）及靶向beclin 1的小干扰RNA（small interfering RNA, siRNA）等抑制剂可阻断自噬，使慢性髓系白血病细胞对TMQ0153更为敏感，并增强坏死性凋亡的发生。值得注意的是，TMQ0153诱导的坏死可导致细胞表面钙网蛋白（calreticulin, CRT）与内质网蛋白57（ERp57）暴露，同时释放细胞外ATP与高迁移率族蛋白B1（high mobility group box 1, HMGB1），证实TMQ0153可诱导具有免疫学特征的细胞死亡。我们通过斑马鱼体内抑制K562微瘤形成的实验，验证了TMQ0153的抗癌潜力。综上，本研究结果表明，TMQ0153通过调控细胞应激与氧化还原状态，以浓度依赖性方式引发慢性髓系白血病细胞模型中多种不同的细胞死亡方式，包括程序性坏死。