Lomeguatrib 是一种 O6-甲基鸟嘌呤-DNA-甲基转移酶 (MGMT) 抑制剂，通过靶向多发性骨髓瘤中的双链 DNA 修复来诱导 DNA 损伤诱导的细胞凋亡,Blood

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Lomeguatrib 是一种 O6-甲基鸟嘌呤-DNA-甲基转移酶 (MGMT) 抑制剂，通过靶向多发性骨髓瘤中的双链 DNA 修复来诱导 DNA 损伤诱导的细胞凋亡
Blood ( IF 21.0 ) Pub Date : 2016-12-02 , DOI: 10.1182/blood.v128.22.2103.2103
Dilara Akcora Yildiz ₁ , Tulin Ozkan ₂ , Yunus Yukselten ₃ , Nezaket Turkel Sesli ₄ , Seyma Ozkanca ₂ , Mehmet Gunduz ₅ , Mehmet Ozen ₂ , Meral Beksac ₆ , Asuman Sunguroglu ₇

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背景/目的：多发性骨髓瘤 (MM) 以终末分化浆细胞在骨髓中的异常积累为特征，由持续的 DNA 损伤和基因组不稳定性促进。O6-甲基鸟嘌呤-DNA-甲基转移酶 (MGMT) 是一种 DNA 修复蛋白，可将烷基从鸟嘌呤转移到其半胱氨酸残基，并且被认为是一种重要的化学抗性因素，尤其是在化学疗法诱导的 DNA 烷基化中。越来越多的证据表明，增强的 DNA 修复能力会刺激癌细胞存活并促进基因组不稳定性。因此，针对包括 MGMT 在内的 DNA 修复活动似乎是治疗 MM 的一种有前途的策略。材料和方法：在我们的研究中，MGMT 表达在 MGUS 中进行评估，使用公开可用的基因表达数据集（Gene Expression Omnibus；GSE47552）将 SMM 和 MM 患者与正常浆细胞进行比较。此外，在人 MM 细胞系（NCI H929、RPMI 8226 和 U266）、三个对照 PBMC 样本、匹配的 CD138+ 骨髓瘤和 CD138- 非致瘤细胞中测定了 MGMT mRNA 和蛋白质水平，这些细胞是从相同 MM 患者的骨髓抽吸物中获得的。新诊断（ND，n=12）和复发/难治性（RR，n=8）]分别通过 RealTime Ready PCR Assay 和 Western blot。人体细胞实验得到了安卡拉大学医学院伦理委员会的批准。在用 Lomeguatrib 处理 MM 细胞 48 小时后，通过 MTT 和流式细胞术评估细胞活力、细胞周期进程和细胞凋亡。通过碱性彗星试验和 ΥH2AX 磷酸化的免疫印迹检测 DNA 损伤水平。结果：在 MM 细胞和正常浆细胞之间以及在 ND CD138+ 和 RR CD138+ 骨髓瘤细胞之间检测到类似的 MGMT 表达。Lomeguatrib 对 MGMT 活性的抑制以剂量依赖性方式降低了 MM 细胞活力并诱导了 MGMT 熟练但 MGMT 缺陷的 MM 细胞凋亡。相比之下，Lomeguatrib 不影响来自三名健康供体的 PBMC 和来自三名 MM 患者的 CD138 非致瘤细胞的细胞活力。此外，Lomeguatrib 扰乱了细胞周期并降低了这些细胞中 G1/S 特异性 CyclinE1 的磷酸化，并导致 DNA 损伤增加。蛋白质印迹分析显示 Fen1 的蛋白质水平降低，Ku-70 和 Rad51 参与双链 DNA 断裂修复。结论：这些结果表明 MGMT 促进 MGMT 熟练 MM 细胞中 DNA 双链断裂的修复，MGMT 抑制增强 DNA 损伤诱导的细胞凋亡，表明 Lomeguatrib 抑制 MGMT 可能是克服 MM 中不忠实 DNA 修复的有用策略。这项研究得到了土耳其科学技术研究委员会 (No:113Z383) 的支持。披露 Beksac：Celgene、Janssen Cilag Amgen、Novartis、Takeda：Honoraria，Speakers Bureau。表明 Lomeguatrib 对 MGMT 的抑制可能是克服 MM 中不忠实 DNA 修复的有用策略。这项研究得到了土耳其科学技术研究委员会 (No:113Z383) 的支持。披露 Beksac：Celgene、Janssen Cilag Amgen、Novartis、Takeda：Honoraria，Speakers Bureau。表明 Lomeguatrib 对 MGMT 的抑制可能是克服 MM 中不忠实 DNA 修复的有用策略。这项研究得到了土耳其科学技术研究委员会 (No:113Z383) 的支持。披露 Beksac：Celgene、Janssen Cilag Amgen、Novartis、Takeda：Honoraria，Speakers Bureau。

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Lomeguatrib, a O6-Methylguanine-DNA-Methyltransferase (MGMT) Inhibitor, Induces DNA Damage Induced Apoptosis By Targeting Double-Strand DNA Repair in Multiple Myeloma

Background/Aim:Multiple Myeloma (MM), characterized by aberrant accumulation of terminally differentiated plasma cells in the bone marrow, is promoted by ongoing DNA damage and genomic instability. O6-methylguanine-DNA-methyltransferase (MGMT), a DNA repair protein, transfers the alkyl group from guanine to its cysteine residue and is implicated as an important chemoresistance factor particularly in chemotherapy-induced DNA alkylation. A growing body of evidence has shown that enhanced DNA repair capacity stimulates cancer cell survival and facilitates genomic instability. Thus, targeting DNA repair activities including MGMT seems to be a promising strategy for the treatment of MM. Materials and Methods: In our study, MGMT expression was evaluated in MGUS, SMM and MM patients compared to normal plasma cells using publically available gene expression data sets (Gene Expression Omnibus; GSE47552). Furthermore, MGMT mRNA and protein levels were determined in human MM cell lines (NCI H929, RPMI 8226 and U266), three samples of control PBMCs, matched CD138+ myeloma and CD138- non-tumorigenic cells obtained from bone marrow aspirates of same MM patients [newly diagnosed (ND, n=12) and relapsed / refractory (RR, n=8)] by RealTime Ready PCR Assay and Western blot, respectively. The experiments with human cells were approved by the Ethical Committee of Ankara University School of Medicine. Following treatment of MM cells with Lomeguatrib for 48 hours, cell viability, cell cycle progression and apoptosis were assessed by MTT and flow cytometry. DNA damage levels were examined by alkaline comet assay and immunoblotting of ΥH2AX phosphorylation. Results: Comparable MGMT expression was detected between MM cells and normal plasma cells, and between ND CD138+ and RR CD138+ myeloma cells. Inhibition of MGMT activity by Lomeguatrib reduced MM cell viability and induced apoptosis in MGMT proficient but not in MGMT deficient MM cells in a dose-dependent manner. In contrast, Lomeguatrib did not affect the cell viability of PBMCs from three healthy donors and CD138- non-tumorigenic cells from three MM patients. Furthermore, Lomeguatrib perturbed cell cycle and decreased phosphorylation of G1/S specific CyclinE1 in these cells and led to an increase in DNA damage. Western blot analysis revealed a reduction in protein levels of Fen1, Ku-70 and Rad51 which are involved in double-strand DNA break repair. Conclusion: These results revealed that MGMT promotes repair of DNA double-strand breaks in MGMT proficient MM cells and MGMT inhibition enhances DNA damage-induced apoptosis, suggesting that inhibition of MGMT by Lomeguatrib might be a useful strategy to overcome unfaithful DNA repair in MM. This research has been supported by The Scientific and Technological Research Council of Turkey (No:113Z383). Disclosures Beksac:Celgene, Janssen Cilag Amgen, Novartis, Takeda: Honoraria, Speakers Bureau.

更新日期：2016-12-02

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