Deciphering the Molecular Pathways Driving p53 Dependent Cell Death

 描述（由申请人提供）：p53基因是最常见的突变肿瘤抑制基因，在超过50%的人类癌症中具有失活突变。其余一半的癌症含有野生型p53，但含有p53失活的额外手段。最近专注于恢复p53的肿瘤抑制程序的癌症疗法包括小分子抑制剂Nutlin-3，其通过抑制p53与其内源性阻遏物MDM 2之间的相互作用来激活休眠的p53。重要的是，这种非遗传毒性的方法将取代毒性化疗治疗，后者通过DNA损伤激活p53而产生有害的副作用。尽管Nutlin-3具有相当大的动力，但其功效目前受到p53反应的异质性的限制，其中大多数癌细胞经历可逆的细胞周期停滞，而不是治疗上有利的凋亡反应。为了改善基于p53的治疗，需要更好地理解p53诱导的细胞凋亡的机制。此外，先前的研究表明，p53的肿瘤抑制功能是依赖于它作为一个转录因子的作用，但没有直接的p53靶点的日期已被确定为至关重要的p53介导的肿瘤抑制。本文的目的是鉴定使p53应答平衡向凋亡倾斜所必需的细胞因子，并确定p53介导的细胞死亡所必需的直接p53转录靶点。为了回答这些问题，将开发以下具体目标：1）鉴定响应Nutlin-3对p53的非遗传毒性激活的p53依赖性细胞凋亡的新介质，2）鉴定p53介导的细胞死亡所必需的直接转录靶标，和3）确定p53依赖性细胞凋亡的新介质对体内肿瘤抑制的影响。在回答这些问题时，一种强大的功能基因组学方法将应用于两种儿科癌细胞系，这两种细胞系已被鉴定为对Nutlin-3具有强烈的凋亡反应。对于鉴定p53依赖性细胞凋亡的新介质的无偏方法，将利用具有全基因组shRNA文库的正选择筛选，并且最佳候选物的验证将清楚地鉴定当敲低时赋予Nutlin治疗抗性的基因。对于细胞死亡所必需的直接p53转录靶标，将进行类似的阳性选择筛选，仅在现在使用独特组成的聚焦P53 TARGET shRNA文库，靶向约300个直接p53靶标。在验证了最佳候选基因后，将对这些基因介导p53依赖性细胞凋亡的机制进行研究。为了将这些发现转化为临床相关性，还将测试所鉴定的凋亡调节剂介导p53依赖性细胞死亡的能力。 肿瘤异种移植（体内）模型。本文的结果将阐明p53介导的细胞死亡所必需的细胞因子，这对于改善p53再激活策略至关重要。一旦有效，这种治疗方案将适用于所有保留野生型p53的癌症，全球近1100万人。

 DESCRIPTION (provided by applicant): The p53 gene is the most commonly mutated tumor suppressor with inactivating mutations in over 50% of all human cancers. The remaining half of cancers contain wild-type p53, but harbor additional means of p53 inactivation. Recent cancer therapies focused on restoring p53's tumor suppressive program include the small molecule inhibitor Nutlin-3, which activates dormant p53 through inhibiting the interaction between p53 and its endogenous repressor MDM2. Importantly, this non-genotoxic approach would replace toxic chemotherapy treatments, which carry harmful side-effects by activating p53 through means of DNA damage. Despite the considerable momentum with Nutlin-3, its efficacy is currently limited by the heterogeneity of the p53 response, where most cancer cells undergo reversible cell cycle arrest, as opposed to a therapeutically favorable apoptotic response. In order to improve p53-based therapies, a better understanding of the mechanisms governing p53-induced apoptosis is needed. Additionally, previous studies demonstrate that p53s tumor suppressive function is dependent on its role as a transcription factor, yet no direct p53 target t date has been identified as crucial to p53-mediated tumor suppression. The objectives herein are to identify the cellular factors necessary to tip the balance of the p53 response toward apoptosis, and to determine what direct p53 transcriptional targets are necessary for p53 mediated cell death. To answer these questions the following specific aims will be developed: 1) Identify novel mediators of p53-dependent apoptosis in response to non- genotoxic activation of p53 by Nutlin-3, 2) Identify direct transcriptional targets necessary for p53-mediated cell death, and 3) Determine the impact of novel mediators of p53-dependent apoptosis on tumor suppression in vivo. In answering these questions, a powerful functional genomics approach will be applied in two pediatric cancer cell lines, which have been identified as having strong apoptotic responses to Nutlin-3. For an unbiased approach to identify novel mediators of p53-dependent apoptosis, a positive selection screen with a genome wide shRNA library will be utilized, and validation of top candidates will clearly identify genes that, when knocked down, confer resistance to Nutlin treatment. For direct p53 transcriptional targets essential for cell death, a similar positive selection screen will be conducted, only now with a uniquely comprised focused P53TARGET shRNA library, targeting ~300 direct p53 targets. After validation of top candidates, investigations into the mechanism by which these genes mediate p53-dependent apoptosis will be conducted. To translate these findings into clinical relevance, the identified apoptotic regulators will also be tested for their ability to mediate p53-dependent cell death in a tumor xenograft (in vivo) model. Results herein will illuminate the cellular factors essential for p53-mediated cell death, which is vital for the improvement of p53-reactivation strategies. Once effective, this treatment option will be available to all cancers that retain wild-type p53, nearly11 million people worldwide.