Targeting stabilized mutant p53 protein

描述(申请人提供)：绝大多数的p53突变是DNA结合域的错义突变(突变的p53)，产生具有广泛丧失功能的构象异常蛋白。重要的是，错义突变P53蛋白不仅失去了肿瘤抑制功能，而且经常获得致癌功能增益(GOF)来驱动肿瘤的发生。GOF具有较高的增殖、侵袭、转移能力和化疗耐药，有助于恶性进展。GOF的一个中心特征是几乎所有的突变P53蛋白都表现出大量的结构性稳定，而稳定是 使出浑身解数。目前，约有1100万人患有高度稳定的突变p53基因表达的肿瘤。重要的是，我们的发现表明，突变的p53肿瘤的致癌‘连线’从根本上不同于P53缺失的肿瘤，历史上使用的主要临床模型。我们发现突变的P53癌已经发展成对高水平的突变P53的生存的强烈依赖。因此，它的急性停药在异种移植中会引发强烈的自发细胞毒性。我们发现热休克蛋白90分子伴侣(HSP90)是突变P53蛋白稳定的主要决定因素。这确定了突变p53的急性耗竭是一种非常有前途的临床策略，并且 在基于p53的治疗中，一个潜在的相当迅速的目标。HSP90在癌症组织中高度上调，但在正常组织中不表达。重要的是，HSP90机制通过支持包括突变P53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器。从机制上讲，HSP90通过稳定的复合体(“笼”)保护突变的P53不受其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稳定的突变P53的持续表达来维持其在体内的维持，并探索通过攻击它们所依赖的伴侣支持来破坏突变P53的稳定是否是一种有前景的突变P53特异性抗癌新策略。这是一个非常重要的尚未解决的问题，尚未得到充分的研究，迫切需要更多的关注，具有很高的翻译潜力，以实现真正的临床影响。目的1确定高水平突变型P53的持续表达是否是维持体内已建立的肿瘤所必需的。我们将在小鼠身上进行急性消融的遗传原理验证实验。为此，我们建立了一种新的人源化失活突变P53 KI模型，并将在体内自发和诱导癌症的背景下对其进行测试。目的2探讨HSP90-HDAC6分子伴侣的小分子抑制剂在体内对Mt-P53癌细胞是否具有优先治疗作用。这将在异种移植和两个不同的热点突变p53 Ki小鼠模型中进行广泛测试。我们还将在三项针对肺癌和乳腺癌患者的回顾性/前瞻性临床试验中，测试突变p53是否为基于HSP90i的治疗反应的预测生物标记物。此外，还将对药物作用机制进行因果关系研究。目的3是基于我们发现的突变的p53稳定性和肿瘤相关的HSP27伴侣系统之间的一种新的意外联系。我们将测试HSP27伴侣是否是突变P53稳定的机制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.