Genomic stability at the coding regions of the multidrug transporter gene ABCB1: insights into the development of alternative drug resistance mechanisms in human leukemia cells.
Genomic stability at the coding regions of the multidrug transporter gene ABCB1: insights into the development of alternative drug resistance mechanisms in human leukemia cells.
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DOI：
10.20517/cdr.2020.51
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Sikic BI
中科院分区：
文献类型：
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作者：
Chen KG;Duran GE;Mogul MJ;Wang YC;Ross KL;Jaffrézou JP;Huff LM;Johnson KR;Fojo T;Lacayo NJ;Sikic BI
目的：尽管在逆转临床多药耐药(MDR)方面做了大量工作，但使用小分子抑制剂靶向主要的多药转运蛋白ABCB1/P-糖蛋白(P-gp)并不成功，这可能是由于出现了替代的耐药机制。然而，非特异性P-gp抑制剂环孢素(CsA)在急性髓系白血病(AML)患者中显示出显著的临床益处，这可能是使用几代MDR抑制剂的唯一原则验证临床试验。然而，由于缺乏CsA相关的细胞模型，可能导致CsA不成功地调节MDR的突变机制尚未阐明。在这项研究中，我们的目标是建立CsA耐药的白血病模型，并检查这些模型以及包括AML在内的不同类型的人类癌症样本中ABCB1外显子突变的存在或不存在。方法：采用逐步药物共选法建立耐药细胞系，通过药敏试验、罗丹明-123蓄积、[3 H]标记药物输出、ABCB1基因测序、核糖核酸酶保护试验等方法进行鉴定。通过外显子序列分析ABCB1编码区在人群中的变异等位基因频率，评估其基因组稳定性。此外，在不同类型的癌症样本中进一步评估了ABCB1的突变谱，包括国家癌症研究所癌症基因组图谱(TCGA)中的AML。结果：我们报道了两种红白血病变异体RVC和RDC，它们分别与依托泊苷-CsA和阿霉素-CsA药物组合在K562/R7耐药细胞中逐步共筛选而来。有趣的是，保留P-gp表达的RVC和RDC细胞株都表现出对CsA调节耐药的多药耐药表型改变。值得注意的是，即使在长期严格的药物选择下，这些变异细胞中的ABCB1编码区也没有发现突变。在基因组学上，ABCB1在人类群体中表现出相对较低的变异等位基因频率，与几个ABC超家族成员相比。此外，与所有类型的人类癌症相比，ABCB1在AML中的突变频率也非常低。此外，我们发现CsA通过诱导低水平和不稳定的耐药来破坏高耐药细胞的选择。结论：我们的数据表明ABCB1编码区在基因组上是稳定的，并且对药物诱导的突变相对耐药。非ABCB1突变机制是RVC和RDC细胞株耐药表型的原因，这在临床AML患者中也很常见。因此，我们提出了几个相关的模型来解释在没有ABCB1突变的情况下替代耐药机制的发展。
Aim: Despite considerable efforts to reverse clinical multidrug resistance (MDR), targeting the predominant multidrug transporter ABCB1/P-glycoprotein (P-gp) using small molecule inhibitors has been unsuccessful, possibly due to the emergence of alternative drug resistance mechanisms. However, the non-specific P-gp inhibitor cyclosporine (CsA) showed significant clinical benefits in patients with acute myeloid leukemia (AML), which likely represents the only proof-of-principle clinical trial using several generations of MDR inhibitors. Nevertheless, the mutational mechanisms that may underlie unsuccessful MDR modulation by CsA are not elucidated because of the absence of CsA-relevant cellular models. In this study, our aims were to establish CsA-resistant leukemia models and to examine the presence or absence of ABCB1 exonic mutations in these models as well as in diverse types of human cancer samples including AMLs. Methods: Drug-resistant lines were established by stepwise drug co-selection and characterized by drug sensitivity assay, rhodamine-123 accumulation, [3H]-labeled drug export, ABCB1 cDNA sequencing, and RNase protection assay. The genomic stability of the ABCB1 coding regions was evaluated by exome sequencing analysis of variant allele frequencies in human populations. Moreover, the mutational spectrum of ABCB1 was further assessed in diverse types of cancer samples including AMLs in the Cancer Genome Atlas (TCGA) at the National Cancer Institute. Results: We report the development of two erythroleukemia variants, RVC and RDC, which were derived by stepwise co-selection of K562/R7 drug-resistant leukemia cells with the etoposide-CsA and doxorubicin-CsA drug combinations, respectively. Interestingly, both RVC and RDC cell lines, which retained P-gp expression, showed altered multidrug-resistant phenotypes that were resistant to CsA modulation. Strikingly, no mutations were found in the ABCB1 coding regions in these variant cells even under long-term stringent drug selection. Genomically, ABCB1 displayed relatively low variant allele frequencies in human populations when compared with several ABC superfamily members. Moreover, ABCB1 also exhibited a very low mutational frequency in AMLs compared with all types of human cancer. In addition, we found that CsA played a role in undermining the selection of highly drug-resistant cells via induction of low-level and unstable drug resistance. Conclusion: Our data indicate that ABCB1 coding regions are genomically stable and relatively resistant to drug-induced mutations. Non-ABCB1 mutational mechanisms are responsible for the drug-resistant phenotypes in both RVC and RDC cell lines, which are also prevalent in clinical AML patients. Accordingly, we propose several relevant models that account for the development of alternative drug resistance mechanisms in the absence of ABCB1 mutations.