Synergistic antitumor effects of 9.2.27-PE38KDEL and ABT-737 in primary and metastatic brain tumors

Standard treatment, unfortunately, yields a poor prognosis for patients with primary or metastatic cancers in the central nervous system, indicating a necessity for novel therapeutic agents. Immunotoxins (ITs) are a class of promising therapeutic candidates produced by fusing antibody fragments with toxin moieties. In this study, we investigated if inherent resistance to IT cytotoxicity can be overcome by rational combination with pro-apoptotic enhancers. Therefore, we combined ITs (9.2.27-PE38KDEL or Mel-14-PE38KDEL) targeting chondroitin sulfate proteoglycan 4 (CSPG4) with a panel of Bcl-2 family inhibitors (ABT-737, ABT-263, ABT-199 [Venetoclax], A-1155463, and S63845) against patient-derived glioblastoma, melanoma, and breast cancer cells/cell lines. In vitro cytotoxicity assays demonstrated that the addition of the ABT compounds, specifically ABT-737, sensitized the different tumors to IT treatment, and improved the IC50 values of 9.2.27-PE38KDEL up to >1,000-fold. Mechanistic studies using 9.2.27-PE38KDEL and ABT-737 revealed that increased levels of intracellular IT, processed (active) exotoxin, and PARP cleavage correlated with the enhanced sensitivity to the combination treatment. Furthermore, we confirmed the synergistic effect of 9.2.27-PE38KDEL and ABT-737 combination therapy in orthotopic GBM xenograft and cerebral melanoma metastasis models in nude mice. Our study defines strategies for overcoming IT resistance and enhancing specific antitumor cytotoxicity in primary and metastatic brain tumors.

遗憾的是，针对中枢神经系统原发性或转移性癌症患者的标准治疗往往预后不佳，这凸显了开发新型治疗药物的迫切需求。免疫毒素（Immunotoxins, ITs）是一类极具潜力的治疗候选药物，通过将抗体片段与毒素结构域融合制备而成。本研究旨在探究：通过与促凋亡增强剂进行合理联合，能否克服肿瘤细胞对免疫毒素细胞毒性的固有耐药性。为此，我们将靶向硫酸软骨素蛋白聚糖4（chondroitin sulfate proteoglycan 4, CSPG4）的免疫毒素（9.2.27-PE38KDEL或Mel-14-PE38KDEL），与一系列Bcl-2家族抑制剂（ABT-737、ABT-263、ABT-199 [维奈克拉（Venetoclax）]、A-1155463及S63845）联合，针对患者来源的胶质母细胞瘤、黑色素瘤及乳腺癌细胞/细胞系开展实验。体外细胞毒性实验结果显示，加入ABT类化合物（尤其是ABT-737）可使多种肿瘤细胞对免疫毒素治疗增敏，将9.2.27-PE38KDEL的半数抑制浓度（IC50）提升至原水平的1000倍以上。采用9.2.27-PE38KDEL与ABT-737进行的机制研究显示，细胞内免疫毒素含量、加工后（活化）的外毒素水平以及PARP剪切程度的升高，均与联合治疗的增敏效果呈正相关。此外，我们在裸鼠原位胶质母细胞瘤异种移植模型及脑内黑色素瘤转移模型中，验证了9.2.27-PE38KDEL与ABT-737联合治疗的协同抗肿瘤效应。本研究明确了克服中枢神经系统原发性及转移性肿瘤免疫毒素耐药、增强特异性抗肿瘤细胞毒性的可行策略。