Targeting stabilized mutant p53 protein

描述（由申请人提供）：绝大多数 p53 突变是 DNA 结合结构域 (mutp53) 中的错义突变，产生具有广泛废除功能的构象异常蛋白。重要的是，错义 mutp53 蛋白不仅失去其肿瘤抑制功能，而且常常获得致癌功能获得（GOF）来驱动肿瘤发生。 GOF 具有较高的增殖、侵袭、转移能力和化疗耐药性，有助于恶性进展。 GOF 的一个核心特征是几乎所有 mutp53 蛋白都表现出大规模的本构稳定性，而这种稳定性是 发挥GOF。目前，约有 1100 万人患有表达高度稳定的 mutp53 的肿瘤。重要的是，我们的研究结果表明，mutp53 肿瘤的致癌“线路”与 p53 缺失肿瘤有根本不同，p53 缺失肿瘤是历史上使用的首要临床前模型。我们发现 mutp53 癌症的生存对高水平的 mutp53 具有很强的依赖性。因此，其急性戒断会在异种移植物中引发强烈的自发细胞毒性。我们发现热休克蛋白 90 伴侣 (HSP90) 是 mutp53 稳定的主要决定因素。这表明 mutp53 的急性耗竭是一种非常有前途的临床策略，并且 基于 p53 的治疗中可能相当快地实现的目标。 HSP90 在癌症中高度上调，但在正常组织中则不然。重要的是，HSP90 机制通过支持包括 mutp53 在内的构象异常癌基因的正确折叠，成为癌细胞存活的重要促进者。从机制上讲，HSP90 通过稳定复合物（“笼蔽”）保护 mutp53 免受其 E3 连接酶的影响。因此，该项目的目标是确定已建立的肿瘤是否需要持续表达稳定的 mutp53 才能在体内维持，并探索通过攻击它们所依赖的分子伴侣支持来破坏 mutp53 的稳定性是否是一种有前途的新型 mutp53 特异性抗癌策略。这是一个非常重要的未解决问题，尚未得到充分研究，迫切需要更多关注，具有实现真正临床影响的巨大转化潜力。目标 1 确定高水平突变 p53 的持续表达是否对于体内已形成肿瘤的维持至关重要。我们将使用小鼠急性消融进行遗传原理验证实验。为此，我们生成了一种新型人源化不可激活 mutp53 KI 模型，并将在体内自发和诱导癌症环境中对其进行测试。目标 2 询问 HSP90-HDAC6 伴侣的小分子抑制剂是否在体内 mutp53 癌细胞中具有优先治疗反应。这将在异种移植物和两种不同的热点 mutp53 KI 小鼠模型中进行广泛测试。我们还将在肺癌和乳腺癌患者的三项回顾性/前瞻性临床试验中测试 mutp53 是否是对基于 HSP90i 的治疗反应的预测生物标志物。此外，还将进行药物作用机制的因果关系研究。目标 3 基于我们发现的异常 mutp53 稳定性与肿瘤相关 HSP27 伴侣系统之间的一种新的、未被怀疑的联系。我们将测试 HSP27 伴侣是否是 mutp53 稳定的机械独立的第二决定因素。

DESCRIPTION (provided by applicant): The vast majority of p53 mutations are missense mutations in the DNA-binding domain (mutp53) that generate conformationally aberrant proteins with broadly abrogated functions. Importantly, missense mutp53 proteins not only lose their tumor suppressor function, but often acquire oncogenic gain-of-function (GOF) to drive tumorigenesis. GOF contributes to malignant progression with higher proliferation, invasion, metastatic ability and chemoresistance. A central feature of GOF is that nearly all mutp53 proteins exhibit massive constitutive stabilization, and that stabilization is the prerequisite for exerting GOF. Currently, ~ 11 million people are living with tumors expressing highly stabilized mutp53. Importantly, our findings indicate that the oncogenic 'wiring' of mutp53 tumors fundamentally differs from p53-null tumors, historically the premier preclinical model used. We find that mutp53 cancers have developed a strong dependency on high levels of mutp53 for survival. Thus, its acute withdrawal triggers strong spontaneous cytotoxicity in xenografts. We identified that the heat shock protein 90 chaperone (HSP90) is a major determinant of mutp53 stabilization. This identifies acute depletion of mutp53 as a very promising clinical strategy, and a potentially rather rapidly attainable goal in p53-based therapy. HSP90 is highly upregulated specifically in cancers but not in normal tissues. Importantly, the HSP90 machinery is a crucial facilitator of cancer cell survival by supporting proper folding of conformationally aberrant oncogenes including mutp53. Mechanistically, HSP90 protects mutp53 via stable complex ('caging') from its E3 ligases. Thus, goals of this project are to determine whether established tumors require continued expression of stabilized mutp53 for their maintenance in vivo, and to explore whether destabilizing mutp53 - by attacking the chaperone support on which they depend - is a promising new mutp53-specific anticancer strategy. This is an immensely important unsolved problem that is understudied and urgently needs more attention, with a high translational potential to achieve true clinical impact. Aim 1 determines if continued expression of high levels of mutant p53 is essential for maintenance of established tumors in vivo. We will perform genetic proof-of-principle experiments using acute ablation in mice. To this end, we generated a novel humanized inactivatable mutp53 KI model and will test it in a spontaneous and induced cancer context in vivo. Aim 2 asks whether small molecule inhibitors of the HSP90-HDAC6 chaperone have a preferential therapeutic response in mutp53 cancer cells in vivo. This will be extensively tested in xenografts and two different hotspot mutp53 KI mouse models. We will also test if mutp53 is a predictive biomarker for response to HSP90i-based therapy in three retrospective/ prospective clinical trials of lung and breast cancer patients. Also, causality studies on mechanism of drug action will be performed. Aim 3 is based on a novel unsuspected link between aberrant mutp53 stabilization and the tumor-associated HSP27 chaperone system that we uncovered. We will test if the HSP27 chaperone is a mechanistically independent second determinant of mutp53 stabilization.