背景急性髓性白血病(AML)是一种侵袭性血液系统恶性肿瘤,其特征是由白血病干细胞(LSCs)引发的未成熟白血病成髓细胞的积累,LSCs也被认为是化疗耐药的根本原因。重新使用强心苷治疗癌症已获得越来越多的关注和支持证据,但是强心苷如何有效地靶向LSCs,例如,是否涉及细胞分化,在很大程度上仍未探索。
方法:地高辛,用户设计的洋地黄毒苷-α-L-鼠李糖苷(D6-MA),并对具有不同成熟表型的各种人AML来源的细胞进行了测试。在这里,我们建立了两个研究模型来专门确定强心苷对LSC死亡和分化的影响-一个允许LSCs和白血病祖细胞(LPCs)动力学的变化,而另一个人保持着未分化的地位。研究了强心苷诱导的细胞毒性的调节机制,并与细胞周期分布和凋亡机制有关。
结果:含有CD34+LSCs/LPCs的原始AML细胞对纳摩尔浓度的强心苷非常敏感,ouabain表现出最大的效率。Ouabain优先诱导LSCs的caspase依赖性凋亡,独立于其细胞分化状态,如(i)在获得小于15%分化的AML细胞中通过哇巴因显著诱导凋亡和(ii)在富集的LSCs中比在LPCs中更高的凋亡率所证明的。我们根据细胞周期分布将LSCs和LPCs分为G0/G1、和G2/M细胞,并揭示了LSCs中的G0/G1细胞,这是它的主要亚群,是乌班最高的响应者,这表明LSCs和LPCs之间的哇巴因敏感性差异涉及不同的细胞周期分布和内在的凋亡调节机制。Further,Mcl-1和c-Myc,在LSCs和LPCs中差异表达,被发现是决定在AML细胞中哇巴因敏感性的关键凋亡介质。通过部分涉及抑制Mcl-1蛋白合成和诱导c-Myc降解的机制,Ouabain在LSC中比在LPC中诱导Mcl-1和c-Myc更快的损失。
结论:我们的数据为通过不同的细胞周期和凋亡机制靶向LSCs,重新利用强心苷治疗复发/难治性AML提供了新的见解。视频摘要。
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by an accumulation of immature leukemic myeloblasts initiating from leukemic stem cells (LSCs)-the subpopulation that is also considered the root cause of chemotherapy resistance. Repurposing cardiac glycosides to treat cancers has gained increasing attention and supporting evidence, but how cardiac glycosides effectively target LSCs, e.g., whether it involves cell differentiation, remains largely unexplored.
Digoxin, a user-designed digitoxigenin-α-L-rhamnoside (D6-MA), and ouabain were tested against various human AML-derived cells with different maturation phenotypes. Herein, we established two study models to specifically determine the effects of cardiac glycosides on LSC death and differentiation-one allowed change in dynamics of LSCs and leukemic progenitor cells (LPCs), while another maintained their undifferentiated status. Regulatory mechanisms underlying cardiac glycoside-induced cytotoxicity were investigated and linked to cell cycle distribution and apoptotic machinery.
Primitive AML cells containing CD34+ LSCs/LPCs were very responsive to nanomolar concentrations of cardiac glycosides, with ouabain showing the greatest efficiency. Ouabain preferentially induces caspase-dependent apoptosis in LSCs, independent of its cell differentiation status, as evidenced by (i) the tremendous induction of apoptosis by ouabain in AML cells that acquired less than 15% differentiation and (ii) the higher rate of apoptosis in enriched LSCs than in LPCs. We sorted LSCs and LPCs according to their cell cycle distribution into G0/G1, S, and G2/M cells and revealed that G0/G1 cells in LSCs, which was its major subpopulation, were the top ouabain responders, indicating that the difference in ouabain sensitivity between LSCs and LPCs involved both distinct cell cycle distribution and intrinsic apoptosis regulatory mechanisms. Further, Mcl-1 and c-Myc, which were differentially expressed in LSCs and LPCs, were found to be the key apoptosis mediators that determined ouabain sensitivity in AML cells. Ouabain induces a more rapid loss of Mcl-1 and c-Myc in LSCs than in LPCs via the mechanisms that in part involve an inhibition of Mcl-1 protein synthesis and an induction of c-Myc degradation.
Our data provide new insight for repurposing cardiac glycosides for the treatment of relapsed/refractory AML through targeting LSCs via distinct cell cycle and apoptosis machinery. Video Abstract.