Purine Analog-Like Properties of Bendamustine Underlie Rapid Activation of DNA Damage Response and Synergistic Effects with Pyrimidine Analogues in Lymphoid Malignancies

Bendamustine has shown considerable clinical activity against indolent lymphoid malignancies as a single agent or in combination with rituximab, but combination with additional anti-cancer drugs may be required for refractory and/or relapsed cases as well as other intractable tumors. In this study, we attempted to determine suitable anti-cancer drugs to be combined with bendamustine for the treatment of mantle cell lymphoma, diffuse large B-cell lymphoma, aggressive lymphomas and multiple myeloma, all of which are relatively resistant to this drug, and investigated the mechanisms underlying synergism. Isobologram analysis revealed that bendamustine had synergistic effects with alkylating agents (4-hydroperoxy-cyclophosphamide, chlorambucil and melphalan) and pyrimidine analogues (cytosine arabinoside, gemcitabine and decitabine) in HBL-2, B104, Namalwa and U266 cell lines, which represent the above entities respectively. In cell cycle analysis, bendamustine induced late S-phase arrest, which was enhanced by 4-hydroperoxy-cyclophosphamide, and potentiated early S-phase arrest by cytosine arabinoside (Ara-C), followed by a robust increase in the size of sub-G1 fractions. Bendamustine was able to elicit DNA damage response and subsequent apoptosis faster and with shorter exposure than other alkylating agents due to rapid intracellular incorporation via equilibrative nucleoside transporters (ENTs). Furthermore, bendamustine increased the expression of ENT1 at both mRNA and protein levels and enhanced the uptake of Ara-C and subsequent increase in Ara-C triphosphate (Ara-CTP) in HBL-2 cells to an extent comparable with the purine analog fludarabine. These purine analog-like properties of bendamustine may underlie favorable combinations with other alkylators and pyrimidine analogues. Our findings may provide a theoretical basis for the development of more effective bendamustine-based combination therapies.

苯达莫司汀（Bendamustine）作为单药或与利妥昔单抗（rituximab）联合应用时，对惰性淋巴系恶性肿瘤展现出显著的临床活性，但针对难治性、复发性病例及其他难治性肿瘤，往往需要联合其他抗肿瘤药物方能奏效。本研究旨在筛选适配苯达莫司汀的联合抗肿瘤药物，用于治疗套细胞淋巴瘤、弥漫大B细胞淋巴瘤、侵袭性淋巴瘤及多发性骨髓瘤——上述肿瘤均对苯达莫司汀存在相对耐药性——同时探究二者协同作用的潜在分子机制。等效线分析法（Isobologram analysis）结果显示，在分别对应上述肿瘤亚型的HBL-2、B104、Namalwa及U266细胞系中，苯达莫司汀可与烷化剂（4-过氧环磷酰胺、苯丁酸氮芥与美法仑）及嘧啶类似物（阿糖胞苷、吉西他滨与地西他滨）产生协同抗肿瘤效应。细胞周期分析结果表明，苯达莫司汀可诱导细胞出现晚期S期阻滞，该效应可被4-过氧环磷酰胺进一步增强；而阿糖胞苷（Ara-C）则可强化苯达莫司汀介导的早期S期阻滞，后续伴随亚G1峰比例的显著升高。相较于其他烷化剂，苯达莫司汀可通过平衡型核苷转运蛋白（ENTs）快速被细胞摄取，因此能在更短的暴露时长内，更快激活DNA损伤应答通路并诱导细胞凋亡。此外，苯达莫司汀可在信使RNA（mRNA）及蛋白水平上调平衡型核苷转运蛋白1（ENT1）的表达，同时可提升HBL-2细胞对阿糖胞苷的摄取效率，进而增加阿糖胞苷三磷酸（Ara-CTP）的生成量，其效果与嘌呤类似物氟达拉滨相当。苯达莫司汀所具备的这类类似嘌呤核苷的特性，可能是其与其他烷化剂及嘧啶类似物产生协同作用的核心基础。本研究结果可为开发更高效的苯达莫司汀联合治疗方案提供坚实的理论依据。