Department of Oncology, Barbara Ann Karmanos Cancer Institute (KCI), Detroit, MI 48201, USA.
J Hematol Oncol. 2012 Sep 18;5:57. doi: 10.1186/1756-8722-5-57.
Lymphomas frequently retain wild-type (wt) p53 function but overexpress HDM2, thereby compromising p53 activity. Therefore, lymphoma is a suitable model for studying the therapeutic value of disrupting the HDM2-p53 interaction by small-molecule inhibitors (SMIs). HDM2 have been developed and are under various stages of preclinical and clinical investigation. Previously, we examined the anti-lymphoma activity of MI-319, the laboratory grade of a new class of HDM2 SMI, the spiro-oxindole, in follicular lymphoma. Since then, MI-219, the clinical grade has become readily available. This study further examines the preclinical effects and mechanisms of MI-219 in a panel of human lymphoma cell lines as well as a cohort of patient-derived B-lymphocytes for its potential clinical use.
Preclinical assessment of MI-219 was evaluated by means of an in vitro and ex vivo approach and compared to Nutlin-3, the gold standard. Characterization of p53 activity and stability were assessed by quantitative PCR, Western blot, and immunoprecipitation. Biological outcome was measured using Trypan blue exclusion assay, Annexin V/PI, PARP and caspase-3 cleavage. Surprisingly, the overall biological effects of Nutlin-3 were more delayed (48 h) while MI-219 triggered an earlier response (12-24 h), predominantly in the form of apoptotic cell death. Using a cell free autoubiquitination assay, neither agent interfered with HDM2 E3 ligase function. MI-219 was more effective in upregulating wt-p53 stabilization compared to Nutlin-3. MI-219, but not Nutlin-3, enhanced the autoubiquitination and degradation of HDM2.
Our data reveals unexpected differences between MI-219 and the well-studied Nutlin-3 in lymphoma cell lines and patient samples. We suggest a novel mechanism for MI-219 that alters the functional activity of HDM2 through enhanced autoubiquitination and degradation. Additionally, this mechanism appears to correspond to biological outcome. Our results provide evidence that different classes of HDM2 SMIs elicit molecular events that extend beyond HDM2-p53 dissociation which may be of biological and potentially therapeutic importance.
淋巴瘤通常保留野生型（wt）p53 功能，但过表达 HDM2，从而降低 p53 活性。因此，淋巴瘤是研究通过小分子抑制剂（SMI）破坏 HDM2-p53 相互作用的治疗价值的合适模型。已经开发出 HDM2，并处于临床前和临床研究的各个阶段。此前，我们研究了新型 HDM2 SMI 螺环-氧吲哚 MI-319 在滤泡性淋巴瘤中的抗淋巴瘤活性。此后，临床级 MI-219 已可获得。本研究进一步在一系列人淋巴瘤细胞系和患者来源的 B 淋巴细胞中研究 MI-219 的临床前作用和机制，以评估其潜在的临床应用。
通过体外和离体方法评估 MI-219 的临床前评估，并与金标准 Nutlin-3 进行比较。通过定量 PCR、Western blot 和免疫沉淀评估 p53 活性和稳定性的特征。使用台盼蓝排斥试验、Annexin V/PI、PARP 和 caspase-3 切割测量生物学结果。令人惊讶的是，Nutlin-3 的整体生物学作用更延迟（48 小时），而 MI-219 则引发更早的反应（12-24 小时），主要以凋亡细胞死亡的形式。使用无细胞自泛素化测定，两种药物均不干扰 HDM2 E3 连接酶功能。与 Nutlin-3 相比，MI-219 更有效地上调 wt-p53 稳定性。MI-219 增强了 HDM2 的自泛素化和降解，而 Nutlin-3 则没有。
我们的数据揭示了 MI-219 和在淋巴瘤细胞系和患者样本中经过充分研究的 Nutlin-3 之间的意外差异。我们提出了一种新的 MI-219 作用机制，该机制通过增强自泛素化和降解来改变 HDM2 的功能活性。此外，这种机制似乎与生物学结果相对应。我们的结果提供了证据，表明不同类别的 HDM2 SMI 引发的分子事件超出了 HDM2-p53 解离，这可能具有生物学和潜在治疗意义。
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