Significant Differences in the Development of Acquired Resistance to the MDM2 Inhibitor SAR405838 between <i>In Vitro</i> and <i>In Vivo</i> Drug Treatment

SAR405838 is a potent and specific MDM2 inhibitor currently being evaluated in Phase I clinical trials for the treatment of human cancer. Using the SJSA-1 osteosarcoma cell line which harbors an amplified MDM2 gene and wild-type p53, we have investigated the acquired resistance mechanisms both in vitro and in vivo to SAR405838. Treatment of SJSA-1 cells with SAR405838 in vitro leads to dose-dependent cell growth inhibition, cell cycle arrest and robust apoptosis. However, prolonged treatment of SJSA-1 cells in vitro with SAR405838 results in profound acquired resistance to the drug. Analysis of in vitro-derived resistant cell lines showed that p53 is mutated in the DNA binding domain and can no longer be activated by SAR405838. Treatment of the parental SJSA-1 xenograft tumors with SAR405838 in mice yields rapid tumor regression but the tumors eventually regrow. Culturing the regrown tumors established a number of sublines, which showed only modest (3–5 times) loss of sensitivity to SAR405838 in vitro. Sequencing of the p53 showed that it retains its wild-type status in these in vivo sublines, with the exception of one subline, which harbors a single heterozygous C176F p53 mutation. Using xenograft models of two in vivo derived sublines, which has either wild-type p53 or p53 containing a single heterozygous C176F mutation, we showed that while SAR405838 effectively achieves partial tumor regression in these models, it no longer induces complete tumor regression and tumors resume growth once the treatment is stopped. Harvesting and culturing tumors obtained from a prolonged treatment with SAR405838 in mice established additional in vivo sublines, which all contain a single heterozygous C176F mutation with no additional p53 mutation detected. Interestingly, SAR405838 can still effectively activate p53 in all sublines containing a single heterozygous C176F mutation, with a moderately reduced potency as compared to that in the parental cell line. Consistently, SAR405838 is 3–5 times less effective in all the in vivo derived sublines containing a single heterozygous C176F p53 mutation than in the SJSA-1 parental cell line in assays of cell growth and apoptosis. Computational modeling suggested that a p53 tetramer containing two wild-type p53 molecules and two C176F mutated molecules can maintain the structural stability and interactions with DNA by formation of additional hydrophobic and cation-π interactions which compensate for the loss of sulphur-zinc coordination. Our data thus show that SJSA-1 tumor cells acquire very different levels of resistance in vitro and in vivo to the MDM2 inhibitor SAR405838. Our present study may have a significant implication for the investigation of resistant mechanisms for other classes of anticancer drugs.SAR405838是一种强效且特异性的MDM2抑制剂（MDM2 inhibitor），目前正处于I期临床试验阶段，用于人类癌症的治疗。本研究采用携带扩增MDM2基因（MDM2 gene）且p53为野生型的SJSA-1骨肉瘤细胞系（SJSA-1 osteosarcoma cell line），从体外与体内两个层面探究了SAR405838的获得性耐药机制。体外使用SAR405838处理SJSA-1细胞，可引发呈剂量依赖性（dose-dependent）的细胞生长抑制、细胞周期阻滞（cell cycle arrest）以及显著的细胞凋亡（apoptosis）。然而，长期体外暴露于SAR405838的SJSA-1细胞会对该药物产生显著的获得性耐药。对体外构建的耐药细胞系进行分析后发现，p53的DNA结合结构域发生突变，无法再被SAR405838激活。向携带亲本SJSA-1异种移植瘤（xenograft tumor）的小鼠施用SAR405838，可快速实现肿瘤消退，但肿瘤最终会复发。对复发肿瘤进行体外培养，获得了多个亚系，这些亚系在体外对SAR405838的敏感性仅出现3~5倍的轻度下降。对这些亚系的p53进行测序发现，除一个亚系携带单个杂合C176F突变型p53（single heterozygous C176F p53 mutation）外，其余亚系的p53均保持野生型状态。随后，我们采用携带野生型p53与携带单个杂合C176F突变型p53的两个体内来源亚系的异种移植模型开展实验，结果显示：SAR405838虽可有效实现部分肿瘤消退，但无法诱导完全肿瘤消退，且停药后肿瘤会恢复生长。从长期接受SAR405838治疗的小鼠体内收获肿瘤并进行体外培养，获得了更多体内亚系，这些亚系均仅携带单个杂合C176F p53突变，未检测到其他p53突变。值得注意的是，在所有携带单个杂合C176F突变的亚系中，SAR405838仍可有效激活p53，仅相较于亲本细胞系，其活性出现适度降低。与此一致的是，在细胞生长与细胞凋亡实验中，所有携带单个杂合C176F p53突变的体内亚系对SAR405838的敏感性均较亲本SJSA-1细胞低3~5倍。计算建模（computational modeling）结果显示，包含2个野生型p53分子与2个C176F突变p53分子的p53四聚体，可通过形成额外的疏水相互作用（hydrophobic interaction）与阳离子-π相互作用（cation-π interaction），维持结构稳定性以及与DNA的结合能力，从而弥补硫-锌配位（sulphur-zinc coordination）缺失带来的影响。综上，SJSA-1肿瘤细胞在体外与体内对MDM2抑制剂SAR405838产生的耐药程度存在显著差异。本研究可为其他类别抗癌药物的耐药机制研究提供重要参考价值。