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（mutp 53）中的错义突变，其产生具有广泛废除功能的构象异常蛋白。重要的是，错义mutp 53蛋白不仅失去了它们的肿瘤抑制功能，而且经常获得致癌功能获得（GOF）来驱动肿瘤发生。GOF导致恶性进展，具有更高的增殖、侵袭、转移能力和化学抗性。GOF的一个中心特征是几乎所有的mutp 53蛋白都表现出大量的组成型稳定性，并且这种稳定性是GOF形成的先决条件。 使用GOF。目前，约有1100万人患有表达高度稳定的mutp 53的肿瘤。重要的是，我们的发现表明mutp 53肿瘤的致癌“布线”从根本上不同于p53-null肿瘤，历史上使用的首要临床前模型。我们发现，mutp 53癌细胞的生存对高水平的mutp 53有很强的依赖性。因此，其急性戒断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（HSP 90）是mutp 53稳定的主要决定因素。这表明急性清除mutp 53是一种非常有前途的临床策略， 这是基于p53的治疗中可能相当快速达到的目标。HSP 90在癌症中特异性地高度上调，但在正常组织中不上调。重要的是，HSP 90机制通过支持包括mutp 53在内的构象异常癌基因的正确折叠，是癌细胞存活的关键促进剂。从机制上讲，HSP 90通过稳定的复合物（“笼”）保护mutp 53免受其E3连接酶的影响。因此，该项目的目标是确定是否建立肿瘤需要稳定的mutp 53的持续表达，以维持其在体内，并探讨是否不稳定的mutp 53-通过攻击分子伴侣的支持，他们依赖-是一个有前途的新的mutp 53特异性抗癌策略。这是一个非常重要的未解决的问题，研究不足，迫切需要更多的关注，具有很高的转化潜力，以实现真正的临床影响。目的1确定高水平的突变型p53的持续表达是否是维持体内已建立的肿瘤所必需的。我们将在小鼠中使用急性消融进行遗传原理验证实验。为此，我们产生了一种新的人源化的失活mutp 53 KI模型，并将在体内自发和诱导的癌症背景下对其进行测试。目的2询问HSP 90-HDAC 6分子伴侣的小分子抑制剂是否在体内mutp 53癌细胞中具有优先的治疗反应。这将在异种移植物和两种不同的热点mutp 53 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mutp 53是否是对基于HSP 90 i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。目的3是基于我们发现的一种新的未知的异常mutp 53稳定性和肿瘤相关的HSP 27分子伴侣系统之间的联系。我们将测试，如果热休克蛋白27分子伴侣是一个机械独立的第二个决定因素mutp 53稳定。

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.