Mutant p53 as actionable cancer-specific target

项目摘要 绝大多数p53突变是DNA结合结构域中的错义突变（称为“mutp 53”）， 产生具有广泛废除功能的构象异常蛋白质。重要的是，在以前的赠款中， 我们建立了新的小鼠模型，明确证明某些热点错义突变体p53 蛋白质不仅失去了它们的肿瘤抑制功能，而且获得了广泛的致癌性功能获得（GOF） 活动（'mutp 53 GOF'）。我们的人源化p53 R248 Q敲入小鼠（称为“Q”小鼠）提供了长期寻求的 所有自发发生的肿瘤类型的更快发作和显著更短的生存期的引人注目的表型 与p53无效同窝出生的小鼠相比。重要的是，我们的发现转化为人类癌症。在Li-Fraumeni患者中 携带p53生殖系突变，Q等位基因显著加速肿瘤发病10.5年，并导致 与p53 null样Li-Fraumeni患者相比，死亡率增加。此外，从TCGA收集的证据 数据表明，携带特异性GOF等位基因的散发性癌症患者的死亡率高于 p53突变在功能上无效GOF导致恶性进展， 增殖、侵袭、转移、化学抗性、基质重塑和重编程代谢。一 GOF的中心特征是mutp 53蛋白表现出大量的组成型稳定性， 这是发挥GOF的前提。我们鉴定了HSP 90分子伴侣机制，它保护mutp 53免受 它的E3泛素连接酶，作为体内稳定的主要决定因素。全球约有1100万人生活在 肿瘤表达高度稳定的mutp 53。重要的是，我们的研究结果表明， mutp 53肿瘤从根本上不同于p53无效肿瘤，p53无效肿瘤在历史上是最重要的临床前模型。 采用值得注意的是，我们确定了本地mutp 53 GOF癌症对 高水平的mutp 53的持续表达用于肿瘤生长、维持和转移。因此，委员会认为， mutp 53的急性遗传（通过HSP 90抑制剂）或药理学（通过HSP 90抑制剂）消融引发强烈的肿瘤 在两种不同的GOF小鼠中的细胞毒性，转化为高达59%的存活率的主要增益，即使在没有细胞毒性的情况下， 野生型p53。这些范式转变的结果确定mutp 53作为一个可行的癌症特异性药物靶点。目的 1到目前为止，我们证明了GOF导致mutp 53肿瘤依赖性-及其治疗利用性-在 淋巴瘤和结直肠癌的背景。我们将评估以mutp 53为靶点的治疗潜力， 其他主要肿瘤类型，特别是肝癌和胰腺癌的上皮来源模型。目标2将 确定体内核心网络的途径和相互作用的合作伙伴介导mutp 53 GOF。我们将使用 ChIPseq/RNAseq和功能蛋白质组分析，直接观察p53发生的动态事件 原发性mutp 53 GOF驱动的淋巴瘤转化过程中的突变。Aim 3将使用全基因组 通过CRISPR介导的基因抑制和激活进行转录干扰，以鉴定关键的遗传学特征。 作为对mutp 53 GOF癌细胞特异性的新治疗靶点的敏化剂。

Project Summary The vast majority of p53 mutations are missense mutations in the DNA-binding domain (termed ‘mutp53’) that generate conformationally aberrant proteins with broadly abrogated functions. Importantly, in the previous grant cycle we generated new mouse models that definitively proved that certain hotspot missense mutant p53 proteins not only lose their tumor suppressor function, but acquire broad oncogenic gain-of-function (GOF) activities (‘mutp53GOF’). Our humanized p53R248Q knockin mice (termed ’Q’ mice) provided the long-sought compelling phenotype of faster onset of all spontaneously arising tumor types and significantly shorter survival compared to p53null littermates. Importantly, our finding translates to human cancers. In Li-Fraumeni patients harboring p53 germline mutations, the Q allele dramatically accelerates tumor onset by 10.5 years and leads to increased mortality compared to p53null-like Li-Fraumeni patients. Moreover, accumulating evidence from TCGA data suggests that sporadic cancer patients harboring specific GOF alleles have higher death rates than patients with p53 mutations that are functionally null. GOF contributes to malignant progression with increased proliferation, invasion, metastasis, chemoresistance, stroma remodeling and reprogrammed metabolism. A central feature of GOF is that mutp53 proteins exhibit massive constitutive stabilization, and that stabilization is the prerequisite for exerting GOF. We identified the HSP90 chaperone machinery, which protects mutp53 from its E3 ubiquitin ligases, as a major determinant of stabilization in vivo. Globally about 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 p53null tumors, which historically was the premier preclinical model used. Notably, we established that autochthonous mutp53GOF cancers develop a strong dependency on continued expression of high levels of mutp53 for tumor growth, maintenance and metastasis. Consequently, acute genetic (via floxQ) or pharmacologic (via Hsp90 inhibitors) ablation of mutp53 triggers strong tumor cytotoxicity in two distinct GOF mice, translating to major gains in survival by up to 59%, even in the absence of wildtype p53. These paradigm-shifting results identify mutp53 as an actionable cancer-specific drug target. Aim 1 So far we demonstrated GOF resulting in mutp53 tumor dependency - and its therapeutic exploitability - in the context of lymphoma and colorectal carcinoma. We will evaluate the therapeutic potential of targeting mutp53 in other major tumor types, specifically in epithelial-derived models of liver and pancreatic carcinomas. Aim 2 will determine the in vivo core network of pathways and interaction partners mediating mutp53GOF. We will use ChIPseq/RNAseq and functional proteome analyses to directly observe the dynamic events that occur upon p53 mutation during transformation in primary mutp53GOF-driven lymphoma. Aim 3 will use genome-wide transcriptional perturbation via CRISPR-mediated gene repression and activation to identify critical genetic sensitizers as novel therapeutic targets specific for mutp53GOF cancer cells.