急性髓系白血病细胞中内在和获得性核苷类似物耐药性的差异。
Differences between intrinsic and acquired nucleoside analogue resistance in acute myeloid leukaemia cells.
机构信息
Institute for Medical Virology, Goethe-University, Frankfurt am Main, Germany.
Faculty of Biological Sciences, Goethe-University, Frankfurt am Main, Germany.
出版信息
J Exp Clin Cancer Res. 2021 Oct 12;40(1):317. doi: 10.1186/s13046-021-02093-4.
BACKGROUND
SAMHD1 mediates resistance to anti-cancer nucleoside analogues, including cytarabine, decitabine, and nelarabine that are commonly used for the treatment of leukaemia, through cleavage of their triphosphorylated forms. Hence, SAMHD1 inhibitors are promising candidates for the sensitisation of leukaemia cells to nucleoside analogue-based therapy. Here, we investigated the effects of the cytosine analogue CNDAC, which has been proposed to be a SAMHD1 inhibitor, in the context of SAMHD1.
METHODS
CNDAC was tested in 13 acute myeloid leukaemia (AML) cell lines, in 26 acute lymphoblastic leukaemia (ALL) cell lines, ten AML sublines adapted to various antileukaemic drugs, 24 single cell-derived clonal AML sublines, and primary leukaemic blasts from 24 AML patients. Moreover, 24 CNDAC-resistant sublines of the AML cell lines HL-60 and PL-21 were established. The SAMHD1 gene was disrupted using CRISPR/Cas9 and SAMHD1 depleted using RNAi, and the viral Vpx protein. Forced DCK expression was achieved by lentiviral transduction. SAMHD1 promoter methylation was determined by PCR after treatment of genomic DNA with the methylation-sensitive HpaII endonuclease. Nucleoside (analogue) triphosphate levels were determined by LC-MS/MS. CNDAC interaction with SAMHD1 was analysed by an enzymatic assay and by crystallisation.
RESULTS
Although the cytosine analogue CNDAC was anticipated to inhibit SAMHD1, SAMHD1 mediated intrinsic CNDAC resistance in leukaemia cells. Accordingly, SAMHD1 depletion increased CNDAC triphosphate (CNDAC-TP) levels and CNDAC toxicity. Enzymatic assays and crystallisation studies confirmed CNDAC-TP to be a SAMHD1 substrate. In 24 CNDAC-adapted acute myeloid leukaemia (AML) sublines, resistance was driven by DCK (catalyses initial nucleoside phosphorylation) loss. CNDAC-adapted sublines displayed cross-resistance only to other DCK substrates (e.g. cytarabine, decitabine). Cell lines adapted to drugs not affected by DCK or SAMHD1 remained CNDAC sensitive. In cytarabine-adapted AML cells, increased SAMHD1 and reduced DCK levels contributed to cytarabine and CNDAC resistance.
CONCLUSION
Intrinsic and acquired resistance to CNDAC and related nucleoside analogues are driven by different mechanisms. The lack of cross-resistance between SAMHD1/ DCK substrates and non-substrates provides scope for next-line therapies after treatment failure.
背景
SAMHD1 通过切割三磷酸化形式来介导对包括阿糖胞苷、地西他滨和奈拉滨在内的抗癌核苷类似物的耐药性，这些类似物常用于治疗白血病。因此，SAMHD1 抑制剂是提高白血病细胞对基于核苷类似物治疗敏感性的有前途的候选药物。在这里，我们研究了胞嘧啶类似物 CNDAC 的作用，该类似物被提议为 SAMHD1 抑制剂，在 SAMHD1 的背景下进行研究。
方法
在 13 种急性髓系白血病（AML）细胞系、26 种急性淋巴细胞白血病（ALL）细胞系、10 种适应各种抗白血病药物的 AML 亚系、24 种单细胞衍生的 AML 亚系和 24 种 AML 患者的原始白血病细胞中测试了 CNDAC。此外，还建立了 AML 细胞系 HL-60 和 PL-21 的 24 种 CNDAC 耐药亚系。使用 CRISPR/Cas9 破坏 SAMHD1 基因，并使用 RNAi 和病毒 Vpx 蛋白耗尽 SAMHD1。通过慢病毒转导实现强制 DCK 表达。用甲基敏感的 HpaII 内切酶处理基因组 DNA 后，通过 PCR 确定 SAMHD1 启动子甲基化。通过 LC-MS/MS 测定核苷（类似物）三磷酸水平。通过酶测定和结晶分析研究 CNDAC 与 SAMHD1 的相互作用。
结果
尽管胞嘧啶类似物 CNDAC 预计会抑制 SAMHD1，但 SAMHD1 在白血病细胞中介导了内在的 CNDAC 耐药性。因此，SAMHD1 耗尽会增加 CNDAC 三磷酸（CNDAC-TP）水平并增加 CNDAC 毒性。酶测定和结晶研究证实 CNDAC-TP 是 SAMHD1 的底物。在 24 种 CNDAC 适应的急性髓系白血病（AML）亚系中，耐药性是由 DCK（催化初始核苷磷酸化）丧失驱动的。CNDAC 适应的亚系仅对其他 DCK 底物（如阿糖胞苷、地西他滨）表现出交叉耐药性。对不受 DCK 或 SAMHD1 影响的药物适应的细胞系仍然对 CNDAC 敏感。在阿糖胞苷适应的 AML 细胞中，SAMHD1 增加和 DCK 减少导致阿糖胞苷和 CNDAC 耐药。
结论
对 CNDAC 和相关核苷类似物的内在和获得性耐药性是由不同的机制驱动的。SAMHD1/DCK 底物和非底物之间缺乏交叉耐药性为治疗失败后的二线治疗提供了机会。
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