Protein translation control in cancer - Mechanistic dissection, in vivo quantification and therapeutic implications

蛋白质翻译异常见于许多癌症，并通过增强癌基因(如MYC)的表达而促进肿瘤的侵袭性。起始是蛋白质翻译的限速步骤，依赖于小核糖体亚基与各种起始因子和包括5‘帽和RNA结构元件在内的mRNA的复杂相互作用。新的工具，如核糖体足迹与RNA测序相结合，现在可以对受影响的mRNA物种进行详细分析，并发现调节元件。当与全基因组筛选相结合时，可以揭示mRNA结构基序与翻译起始因子和相关蛋白的相互作用，并通过药物操作来开发新的癌症治疗方法。这种即将到来的方法的一个例子是RNA解旋酶eIF4A的小分子抑制剂Silvestrol，它已被证明选择性地抑制含有结构性5‘非翻译区(UTRs)的致癌转录本的翻译，如G-四联体。这种策略的开发和验证将从蛋白质翻译的纵向体内量化中受益匪浅，但目前还没有正电子发射断层扫描(PET)示踪剂被验证用于蛋白质翻译，而且经常使用的氨基酸示踪剂的摄取更表明氨基酸转运蛋白的表达，而不是整合到新生蛋白质中。因此，这项工作旨在合成基于翻译抑制剂嘌呤霉素的PET示踪剂，并开发和验证一种定量测定啮齿动物模型中蛋白质翻译的成像方法。同时，参与这一途径的新因子和序列将在生物检测中得到鉴定。特别是，我们将分析eIF4E扰动对单个mRNAs翻译的影响，以确定有助于翻译启动的结构基序。然后，与含有结构化5‘UTRs的报告系统的机制工作将定义作用模式，全基因组CRISPR/Cas9筛选将阐明新的相互作用伙伴，并可能暗示迄今未知的治疗靶点。一旦非侵入性蛋白质翻译成像得到验证，它将被用于在动物模型中表征现有(西维司酮)和新发现的治疗方法的时程和剂量依赖性。这一建议汇集了主办实验室在信使核糖核酸翻译分析和所需的生化分析和策略方面的专业知识，以及申请人在示踪剂开发和PET成像方面的专业知识，以实现这些目标。

Aberrant protein translation is seen in many cancers and contributes to aggressiveness by enhancing the expression of oncogenes (e.g. MYC). Initiation is the rate-limiting step of protein translation and depends on the complex interplay of the small ribosomal subunit with various initiation factors and the mRNA, including 5’cap and RNA structural elements. Novel tools like ribosome footprinting in combination with RNA sequencing now allow for detailed analysis of affected mRNA species and the discovery of regulatory elements. When combined with genome-wide screens, the interplay of mRNA structural motifs with translation initiation factors and associated proteins can be revealed and pharmacologically manipulated to develop novel cancer therapies. An example of such upcoming approach is the small-molecule inhibitor of the RNA helicase eIF4A, silvestrol, that has been shown to selectively inhibit translation of oncogenic transcripts containing structured 5’ untranslated regions (UTRs) like G-quadruplexes. Development and validation of such strategies would highly benefit from longitudinal in vivo quantification of protein translation, but currently no positron emission tomography (PET) tracer has been validated for protein translation, and the uptake of the frequently-used amino acid tracers rather indicates expression of amino acid transporters instead of incorporation into nascent proteins. Therefore this work aims to synthesize PET tracers based on the translation inhibitor puromycin and to develop and validate an imaging protocol for quantification of protein translation in rodent models. In parallel, novel factors and sequences involved in this pathway will be identified in biological assays. In particular, the influence of eIF4E perturbation on translation of individual mRNAs will be analyzed in order to identify structural motifs assisting translation initiation. Mechanistical work with reporter systems containing structured 5’UTRs will then define the mode of action, and genome-wide CRISPR/Cas9 screens will elucidate novel interaction partners and potentially hint at hitherto unknown therapeutic targets. As soon as non-invasive protein translation imaging has been validated, it will be deployed to characterize the time-course and dose-dependency of existing (silvestrol) and newly identified therapies in animal models. This proposal brings together the expertise of the host lab on mRNA translation analysis and the required biochemical assays and strategies, as well as the expertise of the applicant on tracer development and PET imaging to achieve these goals.